Process of preparing mrna-loaded lipid nanoparticles

ABSTRACT

The present invention provides an improved process for lipid nanoparticle formulation and mRNA encapsulation. In some embodiments, the present invention provides a process of encapsulating messenger RNA (mRNA) in lipid nanoparticles comprising a step of mixing a solution of pre-formed lipid nanoparticles and mRNA at a low concentration.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/724,582, filed Aug. 29, 2018, and U.S. Provisional ApplicationSer. No. 62/725,765, filed Aug. 31, 2018, the contents of each of whichare incorporated herein.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “MRT_2030US_Seq_Listing_ST25.txt”,which was created on Aug. 16, 2019 and is 18 KB in size, are herebyincorporated by reference in its entirety.

BACKGROUND

Messenger RNA therapy (MRT) is becoming an increasingly importantapproach for the treatment of a variety of diseases. MRT involvesadministration of messenger RNA (mRNA) to a patient in need of thetherapy for production of the protein encoded by the mRNA within thepatient's body. Lipid nanoparticles are commonly used to encapsulatemRNA for efficient in vivo delivery of mRNA.

To improve lipid nanoparticle delivery, much effort has focused onidentifying novel lipids or particular lipid compositions that canaffect intracellular delivery and/or expression of mRNA, e.g., invarious types of mammalian tissue, organs and/or cells (e.g., mammalianliver cells). However, these existing approaches are costly, timeconsuming and unpredictable.

SUMMARY OF INVENTION

The present invention provides, among other things, a further improvedprocess for preparing mRNA-loaded lipid nanoparticles (mRNA-LNPs) bymixing pre-formed lipid nanoparticles (LNPs) with mRNA. The invention isbased on the surprising discovery that lowering the concentration of thepre-formed LNPs and/or the mRNA during the mixing step providesunexpected benefits such as avoiding formation of aggregates of LNPsand/or decreasing the size of the lipid nanoparticle, while maintainingthe encapsulation efficiency and mRNA recovery. The present invention isparticularly useful for manufacturing mRNA-LNPs with lower levels ofPEG-modified lipids for therapeutic use.

Thus, in one aspect, the present invention provides a process ofencapsulating messenger RNA (mRNA) in lipid nanoparticles comprising:mixing a solution comprising pre-formed lipid nanoparticles and mRNAsuch that lipid nanoparticles encapsulating mRNA are formed, wherein thepre-formed lipid nanoparticles and/or the mRNA are present in thesolution at a concentration of no greater than 0.5 mg/ml.

In some embodiments, the pre-formed lipid nanoparticles are present at aconcentration no greater than 0.4 mg/ml, 0.3 mg/ml, 0.25 mg/ml, 0.2mg/ml, 0.15 mg/ml, 0.1 mg/ml, 0.05 mg/ml, or 0.01 mg/ml.

In some embodiments, the mRNA is present in the solution at aconcentration of no greater than 0.4 mg/ml, 0.3 mg/ml, 0.25 mg/ml, 0.2mg/ml, 0.15 mg/ml, 0.1 mg/ml, 0.05 mg/ml, or 0.01 mg/ml.

In some embodiments, each of the pre-formed lipid nanoparticles and themRNA are present in the solution at a concentration of no greater than0.5 mg/ml, 0.4 mg/ml, 0.3 mg/ml, 0.25 mg/ml, 0.2 mg/ml, 0.15 mg/ml, 0.1mg/ml, 0.05 mg/ml, or 0.01 mg/ml. In some embodiments, each of thepre-formed lipid nanoparticles and the mRNA are present in the solutionat a concentration of no greater than 0.1 mg/ml. In some embodiments,each of the pre-formed lipid nanoparticles and the mRNA are present inthe solution at a concentration of no greater than 0.05 mg/ml.

In some embodiments, a process according to the present inventionfurther comprises a step of diluting the solution to achieve the desiredconcentration of no greater than 0.5 mg/ml.

In some embodiments, the pre-formed lipid nanoparticles comprise aPEG-modified lipid. In some embodiments, the PEG-modified lipidconstitutes less than 3%, less than 2.5%, less than 2%, less than 1.5%,or less than 1% of total lipids in the lipid nanoparticles.

In some embodiments, the PEG-modified lipid constitutes between 0.1% and3%, or between 0.75% and 2.5%, or between 0.5% and 2% of total lipids inthe lipid nanoparticles.

In some embodiments, the PEG-modified lipid constitutes about 1% oftotal lipids in the lipid nanoparticles.

In some embodiments, the solution comprising pre-formed lipidnanoparticles and mRNA comprises less than 10 mM citrate.

In some embodiments, the solution comprising pre-formed lipidnanoparticles and mRNA comprises less than 25% non-aqueous solvent.

In some embodiments, the process according to the present inventionincludes a step of heating one or more of the solutions (i.e., applyingheat from a heat source to the solution) to a temperature (or tomaintain at a temperature) greater than ambient temperature, the onemore solutions being the solution comprising the pre-formed lipidnanoparticles, the solution comprising the mRNA and the mixed solutioncomprising the lipid nanoparticle encapsulated mRNA. In someembodiments, the process includes the step of heating one or both of themRNA solution and the pre-formed lipid nanoparticle solution, prior tothe mixing step. In some embodiments, the process includes heating oneor more one or more of the solution comprising the pre-formed lipidnanoparticles, the solution comprising the mRNA and the solutioncomprising the lipid nanoparticle encapsulated mRNA, during the mixingstep. In some embodiments, the process includes the step of heating thelipid nanoparticle encapsulated mRNA, after the mixing step. In someembodiments, the temperature to which one or more of the solutions isheated (or at which one or more of the solutions is maintained) is or isgreater than about 30° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60°C., 65° C., or 70° C. In some embodiments, the temperature to which oneor more of the solutions is heated ranges from about 25-70° C., about30-70° C., about 35-70° C., about 40-70° C., about 45-70° C., about50-70° C., or about 60-70° C. In some embodiments, the temperaturegreater than ambient temperature to which one or more of the solutionsis heated is about 65° C.

In some embodiments, the process according to the present inventionincludes maintaining at ambient temperature (i.e., not applying heatfrom a heat source to the solution) one or more of the solutioncomprising the pre-formed lipid nanoparticles, the solution comprisingthe mRNA and the mixed solution comprising the lipid nanoparticleencapsulated mRNA. In some embodiments, the process includes the step ofmaintaining at ambient temperature one or both of the mRNA solution andthe pre-formed lipid nanoparticle solution, prior to the mixing step. Insome embodiments, the process includes maintaining at ambienttemperature one or more one or more of the solution comprising thepre-formed lipid nanoparticles, the solution comprising the mRNA and thesolution comprising the lipid nanoparticle encapsulated mRNA, during themixing step. In some embodiments, the process includes the step ofmaintaining at ambient temperature the lipid nanoparticle encapsulatedmRNA, after the mixing step. In some embodiments, the ambienttemperature at which one or more of the solutions is maintained is or isless than about 35° C., 30° C., 25° C., 20° C., or 16° C. In someembodiments, the ambient temperature at which one or more of thesolutions is maintained ranges from about 15-35° C., about 15-30° C.,about 15-25° C., about 15-20° C., about 20-35° C., about 25-35° C.,about 30-35° C., about 20-30° C., about 25-30° C. or about 20-25° C. Insome embodiments, the ambient temperature at which one or more of thesolutions is maintained is 20-25° C.

In some embodiments, the process according to the present inventionincludes performing at ambient temperature the step of mixing thesolution comprising pre-formed lipid nanoparticles and the solutioncomprising mRNA to form lipid nanoparticles encapsulating mRNA.

In some embodiments, the pre-formed lipid nanoparticles are formed bymixing lipids dissolved in ethanol with an aqueous solution. In someembodiments, the lipids contain one or more cationic lipids, one or morehelper lipids, and one or more PEG lipids. In some embodiments, thelipids also contain one or more cholesterol lipids. The pre-formed lipidnanoparticles are formed by the mixing of those lipids. Accordingly, insome embodiments, the pre-formed lipid nanoparticles comprise one ormore cationic lipids, one or more helper lipids, and one or more PEGlipids. In some embodiments, the pre-formed lipid nanoparticles alsocontain one or more cholesterol lipids.

In some embodiments, the one or more cationic lipids are selected fromthe group consisting of cKK-E12, OF-02, C12-200, MC3, DLinDMA,DLinkC2DMA, ICE (Imidazol-based), HGT5000, HGT5001, HGT4003, DODAC,DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE,CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP,DLinSSDMA, KLin-K-DMA, DLin-K-XTC2-DMA,3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione(Target 23),3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione(Target 24), N1GL, N2GL, V1GL, ccBene, ML7, ribose cationic lipids andcombinations thereof.

In some embodiments, the one or more cationic lipids comprise ccBene. Insome embodiments, the one or more cationic lipids comprise ML7. In someembodiments, the one or more cationic lipids comprise DLinSSDMA.

In some embodiments, the one or more cationic lipids are amino lipids.Amino lipids suitable for use in the invention include those describedin WO2017180917, which is hereby incorporated by reference. Exemplaryaminolipids in WO2017180917 include those described at paragraph [0744]such as DLin-MC3-DMA (MC3),(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608), andCompound 18. Other amino lipids include Compound 2, Compound 23,Compound 27, Compound 10, and Compound 20. Further amino lipids suitablefor use in the invention include those described in WO2017112865, whichis hereby incorporated by reference. Exemplary amino lipids inWO2017112865 include a compound according to one of formulae (I),(Ia1)-(Ia6), (Ib), (II), (IIa), (III), (IIIa), (IV), (17-1), (19-1).(19-11), and (20-1), and compounds of paragraphs [00185], [00201],[0276]. In some embodiments, cationic lipids suitable for use in theinvention include those described in WO2016118725, which is herebyincorporated by reference. Exemplary cationic lipids in WO2016118725include those such as KL22 and KL25. In some embodiments, cationiclipids suitable for use in the invention include those described inWO2016118724, which is hereby incorporated by reference. Exemplarycationic lipids in WO2016118725 include those such as KL10,1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

In some embodiments, the one or more non-cationic lipids are selectedfrom DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)).

In some embodiments, the one or more PEG-modified lipids comprise apoly(ethylene) glycol chain of up to 5 kDa in length covalently attachedto a lipid with alkyl chain(s) of C₆-C₂₀ length.

In some embodiments, the pre-formed lipid nanoparticles are purified bya Tangential Flow Filtration (TFF) process. In some embodiments, greaterthan about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% of the purified nanoparticles have a size less than about150 nm (e.g., less than about 145 nm, about 140 nm, about 135 nm, about130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, about 80nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, orabout 50 nm). In some embodiments, substantially all of the purifiednanoparticles have a size less than 150 nm (e.g., less than about 145nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm,about 90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about65 nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments,greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% ofthe purified nanoparticles have a size ranging from 50-150 nm. In someembodiments, substantially all of the purified nanoparticles have a sizeranging from 50-150 nm. In some embodiments, greater than about 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purifiednanoparticles have a size ranging from 80-150 nm. In some embodiments,substantially all of the purified nanoparticles have a size ranging from80-150 nm.

In some embodiments, a process according to the present inventionresults in an encapsulation rate of greater than about 90%, 95%, 96%,97%, 98%, or 99%. In some embodiments, a process according to thepresent invention results in greater than about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.

In some embodiments, the pre-formed lipid nanoparticles and mRNA aremixed using a pump system. In some embodiments, the pump systemcomprises a pulse-less flow pump. In some embodiments, the pump systemis a gear pump. In some embodiments, a suitable pump is a peristalticpump. In some embodiments, a suitable pump is a centrifugal pump. Insome embodiments, the process using a pump system is performed at largescale. For example, in some embodiments, the process includes usingpumps as described herein to mix a solution of at least about 1 mg, 5mg, 10 mg, 50 mg, 100 mg, 500 mg, or 1000 mg of mRNA with a solution ofpre-formed lipid nanoparticles, to produce mRNA encapsulated in lipidnanoparticles. In some embodiments, the process of mixing mRNA withpre-formed lipid nanoparticles provides a composition according to thepresent invention that contains at least about 1 mg, 5 mg, 10 mg, 50 mg,100 mg, 500 mg, or 1000 mg of encapsulated mRNA.

In some embodiments, the solution comprising pre-formed lipidnanoparticles is mixed at a flow rate ranging from about 25-75ml/minute, about 75-200 ml/minute, about 200-350 ml/minute, about350-500 ml/minute, about 500-650 ml/minute, about 650-850 mi/minute, orabout 850-1000 mi/minute. In some embodiments, the solution comprisingpre-formed lipid nanoparticles is mixed at a flow rate of about 50md/minute, about 100 ml/minute, about 150 ml/minute, about 200ml/minute, about 250 ml/minute, about 300 mi/minute, about 350ml/minute, about 400 ml/minute, about 450 ml/minute, about 500ml/minute, about 550 ml/minute, about 600 ml/minute, about 650ml/minute, about 700 ml/minute, about 750 ml/minute, about 800ml/minute, about 850 ml/minute, about 900 ml/minute, about 950ml/minute, or about 1000 mi/minute.

In some embodiments, the mRNA is mixed in a solution at a flow rateranging from about 25-75 ml/minute, about 75-200 ml/minute, about200-350 mi/minute, about 350-500 ml/minute, about 500-650 m/minute,about 650-850 ml/minute, or about 850-1000 ml/minute. In someembodiments, the mRNA is mixed in a solution at a flow rate of about 50mi/minute, about 100 ml/minute, about 150 ml/minute, about 200ml/minute, about 250 ml/minute, about 300 ml/minute, about 350ml/minute, about 400 ml/minute, about 450 ml/minute, about 500ml/minute, about 550 ml/minute, about 600 ml/minute, about 650ml/minute, about 700 ml/minute, about 750 ml/minute, about 800ml/minute, about 850 ml/minute, about 900 ml/minute, about 950ml/minute, or about 1000 ml/minute.

In some embodiments, a process according to the present inventionincludes a step of first generating pre-formed lipid nanoparticlesolution by mixing a citrate buffer with lipids dissolved in ethanol.

In some embodiments, a process according to the present inventionincludes a step of first generating an mRNA solution by mixing a citratebuffer with an mRNA stock solution. In certain embodiments, a suitablecitrate buffer contains about 10 mM citrate, about 150 mM NaCl, pH ofabout 4.5. In some embodiments, a suitable mRNA stock solution containsthe mRNA at a concentration at or greater than about 1 mg/ml, about 10mg/ml, about 50 mg/ml, or about 100 mg/ml.

In some embodiments, the citrate buffer is mixed at a flow rate rangingbetween about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute,1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, or4800-6000 ml/minute. In some embodiments, the citrate buffer is mixed ata flow rate of about 220 ml/minute, about 600 ml/minute, about 1200ml/minute, about 2400 ml/minute, about 3600 ml/minute, about 4800mi/minute, or about 6000 ml/minute.

In some embodiments, the mRNA stock solution is mixed at a flow rateranging between about 10-30 ml/minute, about 30-60 ml/minute, about60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute,about 360-480 ml/minute, or about 480-600 ml/minute. In someembodiments, the mRNA stock solution is mixed at a flow rate of about 20ml/minute, about 40 ml/minute, about 60 ml/minute, about 80 mi/minute,about 100 ml/minute, about 200 ml/minute, about 300 ml/minute, about 400m/minute, about 500 ml/minute, or about 600 ml/minute.

In some embodiments, the lipid nanoparticles encapsulating mRNA areprepared with the pre-formed lipid nanoparticles by mixing an aqueoussolution containing the mRNA with an aqueous solution containing thepre-formed lipid nanoparticles. In some embodiments, the aqueoussolution containing the mRNA and/or the aqueous solution containing thepre-formed lipid nanoparticles is an aqueous solution comprisingpharmaceutically acceptable excipients, including, but not limited to,one or more of trehalose, sucrose, lactose, and mannitol.

In some embodiments, one or both of a non-aqueous solvent, such asethanol, and citrate are absent (i.e., below detectable levels) from oneor both of the solution containing the mRNA and the solution containingthe pre-formed lipid nanoparticles during the mixing addition of themRNA to the pre-formed lipid nanoparticles. In some embodiments, one orboth of the solution containing the mRNA and the solution containing thepre-formed lipid nanoparticles are buffer exchanged to remove one orboth of non-aqueous solvents, such as ethanol, and citrate prior to themixing addition of the mRNA to the pre-formed lipid nanoparticles. Insome embodiments, one or both of the solution containing the mRNA andthe solution containing the pre-formed lipid nanoparticles include onlyresidual citrate during the mixing addition of mRNA to the pre-formedlipid nanoparticles. In some embodiments, one or both of the solutioncontaining the mRNA and the solution containing the pre-formed lipidnanoparticles include only residual non-aqueous solvent, such asethanol. In some embodiments, one or both of the solution containing themRNA and the solution containing the pre-formed lipid nanoparticlescontains less than about 10 mM (e.g., less than about 9 mM, about 8 mM,about 7 mM, about 6 mM, about 5 mM, about 4 mM, about 3 mM, about 2 mM,or about 1 mM) of citrate present during the addition of mRNA to thepre-formed lipid nanoparticles. In some embodiments, one or both of thesolution containing the mRNA and the solution containing the pre-formedlipid nanoparticles contains less than about 25% (e.g., less than about20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, orabout 1%) of non-aqueous solvents, such as ethanol, present during theaddition of mRNA to the pre-formed lipid nanoparticles. In someembodiments, the solution comprising the lipid nanoparticlesencapsulating mRNA does not require any further downstream processing(e.g., buffer exchange and/or further purification steps) after thepre-formed lipid nanoparticles and mRNA are mixed to form that solution.

In another aspect, the present invention provides a composition of lipidnanoparticles encapsulating mRNA generated by a process describedherein. In some embodiments, a substantial amount of the lipidnanoparticles are pre-formed. In some embodiments, at least 85% (e.g.,at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%) of the lipid nanoparticles are pre-formed. In someembodiments, the present invention provides a composition comprisingpurified lipid nanoparticles, wherein greater than about 90% of thepurified lipid nanoparticles have an individual particle size of lessthan about 150 nm (e.g., less than about 145 nm, about 140 nm, about 135nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm,about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about55 nm, or about 50 nm) and greater than about 70% of the purified lipidnanoparticles encapsulate an mRNA within each individual particle. Insome embodiments, greater than about 95%, 96%, 97%, 98%, or 99% of thepurified lipid nanoparticles have an individual particle size of lessthan about 150 nm (e.g., less than about 145 nm, about 140 nm, about 135nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm,about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about55 nm, or about 50 nm). In some embodiments, substantially all of thepurified lipid nanoparticles have an individual particle size of lessthan about 150 nm (e.g., less than about 145 nm, about 140 nm, about 135nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm,about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm, about55 nm, or about 50 nm). In some embodiments, greater than about 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the purifiednanoparticles have a size ranging from 50-150 nm. In some embodiments,substantially all of the purified nanoparticles have a size ranging from50-150 nm. In some embodiments, greater than about 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% of the purified nanoparticles have a sizeranging from 80-150 nm. In some embodiments, substantially all of thepurified nanoparticles have a size ranging from 80-150 nm.

In some embodiments, greater than about 90%, 95%, 96%, 97%, 98%, or 99%of the purified lipid nanoparticles encapsulate an mRNA within eachindividual particle. In some embodiments, substantially all of thepurified lipid nanoparticles encapsulate an mRNA within each individualparticle. In some embodiments, a composition according to the presentinvention contains at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or1000 mg of encapsulated mRNA.

In some embodiments, a pre-formed lipid nanoparticle comprises one ormore cationic lipids, one or more helper lipids and one or more PEGlipids. In some embodiments, each individual lipid nanoparticle alsocomprises one or more cholesterol based lipids. In some embodiments, theone or more cationic lipids are selected from the group consisting ofcKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE (Imidazol-based),HGT5000, HGT5001, HGT4003, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMAand DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP,DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA,3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione(Target 23),3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione(Target 24), N1GL, N2GL, V1GL and combinations thereof.

In some embodiments, the one or more cationic lipids are amino lipids.Amino lipids suitable for use in the invention include those describedin WO2017180917, which is hereby incorporated by reference. Exemplaryaminolipids in WO2017180917 include those described at paragraph 107441such as DLin-MC3-DMA (MC3),(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608), andCompound 18. Other amino lipids include Compound 2, Compound 23,Compound 27, Compound 10, and Compound 20. Further amino lipids suitablefor use in the invention include those described in WO2017112865, whichis hereby incorporated by reference. Exemplary amino lipids inWO2017112865 include a compound according to one of formulae (I),(Ia1)-(Ia6), (Ib), (II), (IIa), (III), (IIIa), (IV), (17-1), (19-1),(19-11), and (20-1), and compounds of paragraphs [00185], [00201],[0276]. In some embodiments, cationic lipids suitable for use in theinvention include those described in WO2016118725, which is herebyincorporated by reference. Exemplary cationic lipids in WO2016118725include those such as KL22 and KL25. In some embodiments, cationiclipids suitable for use in the invention include those described inWO2016118724, which is hereby incorporated by reference. Exemplarycationic lipids in WO2016118725 include those such as KL10,1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), and KL25.

In some embodiments, the one or more non-cationic lipids are selectedfrom DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine). DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)).

In some embodiments, the one or more cholesterol-based lipids ischolesterol or PEGylated cholesterol. In some embodiments, the one ormore PEG-modified lipids contain a poly(ethylene) glycol chain of up to5 kDa in length covalently attached to a lipid with alkyl chain(s) ofC₆-C₂₀ length.

In some embodiments, the present invention is used to encapsulate mRNAcontaining one or more modified nucleotides. In some embodiments, one ormore nucleotides is modified to a pseudouridine. In some embodiments,one or more nucleotides is modified to a 5-methylcytidine. In someembodiments, the present invention is used to encapsulate mRNA that isunmodified.

In some embodiments, a process according to the present inventionresults in no substantial aggregation of lipid nanoparticles.

In other aspects, the present invention provides compositions comprisingmRNA loaded LNPs prepared using various methods described herein. Insome embodiments, the present invention provides compositions comprisingmRNA loaded LNPs (e.g., with greater than 80%, 90%, 95%, 98% or 99%encapsulation efficiency) with no substantial aggregation of LNPs. Insome embodiments, the mRNA loaded LNPs contain a low level ofPEG-modified lipids (e.g., less than 3%, 2.5%, 2%, 1.5%, 1%, or 0.5% ofthe total lipids in LNPs). The present invention further provides amethod of delivering mRNA for in vivo protein production comprisingadministering into a subject a composition of lipid nanoparticlesencapsulating mRNA generated by the process described herein, whereinthe mRNA encodes one or more protein(s) or peptide(s) of interest.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this disclosure, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Both terms are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description, drawings and claims that follow.It should be understood, however, that the detailed description, thedrawings, and the claims, while indicating embodiments of the presentinvention, are given by way of illustration only, not limitation.Various changes and modifications within the scope of the invention willbecome apparent to those skilled in the art.

Definitions

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Alkyl: As used herein, “alkyl” refers to a radical of a straight-chainor branched saturated hydrocarbon group having from 1 to 20 carbon atoms(“C₁₋₂₀ alkyl”). In some embodiments, an alkyl group has 1 to 3 carbonatoms (“C₁₋₃ alkyl”). Examples of C₁₋₃ alkyl groups include methyl (C₁),ethyl (C₂), n-propyl (C₃), and isopropyl (C₃). In some embodiments, analkyl group has 8 to 12 carbon atoms (“C₈₋₁₂ alkyl”). Examples of C₈₋₁₂alkyl groups include, without limitation, n-octyl (C₈), n-nonyl (C₉),n-decyl (C₁₀), n-undecyl (C₁₁), n-dodecyl (C₁₂) and the like. The prefix“n-” (normal) refers to unbranched alkyl groups. For example, n-C₈ alkylrefers to —(CH₂)₇CH₃, n-C₁₀ alkyl refers to —(CH₂)₉CH₃, etc.

Amino acid: As used herein, term “amino acid.” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an 1-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Delivery: As used herein, the term “delivery” encompasses both local andsystemic delivery. For example, delivery of mRNA encompasses situationsin which an mRNA is delivered to a target tissue and the encoded proteinor peptide is expressed and retained within the target tissue (alsoreferred to as “local distribution” or “local delivery”), and situationsin which an mRNA is delivered to a target tissue and the encoded proteinor peptide is expressed and secreted into patient's circulation system(e.g., serum) and systematically distributed and taken up by othertissues (also referred to as “systemic distribution” or “systemicdelivery).

Efficacy: As used herein, the term “efficacy,” or grammaticalequivalents, refers to an improvement of a biologically relevantendpoint, as related to delivery of mRNA that encodes a relevant proteinor peptide. In some embodiments, the biological endpoint is protectingagainst an ammonium chloride challenge at certain timepoints afteradministration.

Encapsulation: As used herein, the term “encapsulation,” or grammaticalequivalent, refers to the process of confining an individual mRNAmolecule within a nanoparticle.

Expression: As used herein, “expression” of a mRNA refers to translationof an mRNA into a peptide (e.g., an antigen), polypeptide, or protein(e.g., an enzyme) and also can include, as indicated by context, thepost-translational modification of the peptide, polypeptide or fullyassembled protein (e.g., enzyme). In this application, the terms“expression” and “production,” and grammatical equivalent, are usedinter-changeably.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control sample or subject (or multiple controlsamples or subjects) in the absence of the treatment described herein. A“control sample” is a sample subjected to the same conditions as a testsample, except for the test article. A “control subject” is a subjectafflicted with the same form of disease as the subject being treated,who is about the same age as the subject being treated.

Impurities: As used herein, the term “impurities” refers to substancesinside a confined amount of liquid, gas, or solid, which differ from thechemical composition of the target material or compound. Impurities arealso referred to as contaminants.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.).

Local distribution or delivery: As used herein, the terms “localdistribution,” “local delivery,” or grammatical equivalent, refer totissue specific delivery or distribution. Typically, local distributionor delivery requires a peptide or protein (e.g., enzyme) encoded bymRNAs be translated and expressed intracellularly or with limitedsecretion that avoids entering the patient's circulation system.

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)”refers to a polynucleotide that encodes at least one peptide,polypeptide or protein. mRNA as used herein encompasses both modifiedand unmodified RNA. mRNA may contain one or more coding and non-codingregions. mRNA can be purified from natural sources, produced usingrecombinant expression systems and optionally purified, chemicallysynthesized, etc. Where appropriate, e.g., in the case of chemicallysynthesized molecules, mRNA can comprise nucleoside analogs such asanalogs having chemically modified bases or sugars, backbonemodifications, etc. An mRNA sequence is presented in the 5′ to 3′direction unless otherwise indicated. In some embodiments, an mRNA is orcomprises natural nucleosides (e.g., adenosine, guanosine, cytidine,uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine,inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine. C-5propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, 2-thiocytidine, pseudouridine, and5-methylcytidine); chemically modified bases; biologically modifiedbases (e.g., methylated bases); intercalated bases; modified sugars(e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose);and/or modified phosphate groups (e.g., phosphorothioates and5′-N-phosphoramidite linkages).

Nucleic acid: As used herein, the term “nucleic acid.” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into a polynucleotide chain. In some embodiments, a nucleicacid is a compound and/or substance that is or can be incorporated intoa polynucleotide chain via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g., nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to a polynucleotide chain comprising individual nucleicacid residues. In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA and/or cDNA. Furthermore, theterms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleicacid analogs, i.e., analogs having other than a phosphodiester backbone.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. A human includes pre- and post-natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable salt: Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge et al., describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences (1977) 66:1-19. Pharmaceutically acceptable salts of thecompounds of this invention include those derived from suitableinorganic and organic acids and bases. Examples of pharmaceuticallyacceptable, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, oxalic acid, maleic acid, tartaric acid, citricacid, succinic acid or malonic acid or by using other methods used inthe art such as ion exchange. Other pharmaceutically acceptable saltsinclude adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl), salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Further pharmaceutically acceptable salts include salts formed from thequaternization of an amine using an appropriate electrophile, e.g., analkyl halide, to form a quarternized alkylated amino salt.

Potency: As used herein, the term “potency,” or grammatical equivalents,refers to expression of protein(s) or peptide(s) that the mRNA encodesand/or the resulting biological effect.

Salt: As used herein the term “salt” refers to an ionic compound thatdoes or may result from a neutralization reaction between an acid and abase.

Systemic distribution or delivery: As used herein, the terms “systemicdistribution,” “systemic delivery,” or grammatical equivalent, refer toa delivery or distribution mechanism or approach that affect the entirebody or an entire organism. Typically, systemic distribution or deliveryis accomplished via body's circulation system, e.g., blood stream.Compared to the definition of “local distribution or delivery.”

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by a disease to be treated. In some embodiments,target tissues include those tissues that display disease-associatedpathology, symptom, or feature.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

Yield: As used herein, the term “yield” refers to the percentage of mRNArecovered after encapsulation as compared to the total mRNA as startingmaterial. In some embodiments, the term “recovery” is usedinterchangeably with the term “yield”.

DETAILED DESCRIPTION

The present invention provides an improved process for lipidnanoparticle (LNP) formulation and mRNA encapsulation based on mixingpre-formed LNPs and mRNA at a low concentration. In some embodiments,one or both of the pre-formed LNPs and mRNA are mixed for encapsulationat a concentration no greater than 1 mg/ml (e.g., no greater than 0.9mg/ml, no greater than 0.8 mg/ml, no greater than 0.7 mg/ml, no greaterthan 0.6 mg/ml, no greater than 0.5 mg/ml, no greater than 0.4 mg/ml, nogreater than 0.3 mg/ml, no greater than 0.2 mg/ml, no greater than 0.1mg/ml, no greater than 0.09 mg/ml, no greater than 0.08 mg/ml, nogreater than 0.07 mg/ml, no greater than 0.06 mg/ml, no greater than0.05 mg/ml, no greater than 0.04 mg/ml, no greater than 0.03 mg/ml, nogreater than 0.02 mg/ml, or no greater than 0.01 mg/ml).

In some embodiments, the resultant encapsulation efficiencies for thepresent lipid nanoparticle formulation and preparation method are around90%. For the delivery of nucleic acids, achieving high encapsulationefficiencies is critical to attain protection of the drug substance andreduce loss of activity in vivo. In addition, a surprising result forthe lipid nanoparticle formulation prepared by the novel method in thecurrent invention is the significantly higher transfection efficiencyobserved in vitro.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention.

Messenger RNA (mRNA)

The present invention may be used to encapsulate any mRNA. mRNA istypically thought of as the type of RNA that carries information fromDNA to the ribosome. Typically, in eukaryotic organisms, mRNA processingcomprises the addition of a “cap” on the 5′ end, and a “tail” on the 3′end. A typical cap is a 7-methylguanosine cap, which is a guanosine thatis linked through a 5′-5′-triphosphate bond to the first transcribednucleotide. The presence of the cap is important in providing resistanceto nucleases found in most eukaryotic cells. The additional of a tail istypically a polyadenylation event whereby a polyadenylyl moiety is addedto the 3′ end of the mRNA molecule. The presence of this “tail” servesto protect the mRNA from exonuclease degradation. Messenger RNA istranslated by the ribosomes into a series of amino acids that make up aprotein.

mRNAs may be synthesized according to any of a variety of known methods.For example, mRNAs according to the present invention may be synthesizedvia in vitro transcription (IVT). Briefly, IVT is typically performedwith a linear or circular DNA template containing a promoter, a pool ofribonucleotide triphosphates, a buffer system that may include DTT andmagnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6RNA polymerase), DNase 1, pyrophosphatase, and/or RNAse inhibitor. Theexact conditions will vary according to the specific application.

In some embodiments, in vitro synthesized mRNA may be purified beforeformulation and encapsulation to remove undesirable impurities includingvarious enzymes and other reagents used during mRNA synthesis.

The present invention may be used to formulate and encapsulate mRNAs ofa variety of lengths. In some embodiments, the present invention may beused to formulate and encapsulate in vitro synthesized mRNA of orgreater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5kb, 5 kb 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15kb, or 20 kb in length. In some embodiments, the present invention maybe used to formulate and encapsulate in vitro synthesized mRNA rangingfrom about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kbin length.

The present invention may be used to formulate and encapsulate mRNA thatis unmodified or mRNA containing one or more modifications thattypically enhance stability. In some embodiments, modifications areselected from modified nucleotides, modified sugar phosphate backbones,and 5′ and/or 3′ untranslated region.

In some embodiments, modifications of mRNA may include modifications ofthe nucleotides of the RNA. A modified mRNA according to the inventioncan include, for example, backbone modifications, sugar modifications orbase modifications. In some embodiments, mRNAs may be synthesized fromnaturally occurring nucleotides and/or nucleotide analogues (modifiednucleotides) including, but not limited to, purines (adenine (A),guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), andas modified nucleotides analogues or derivatives of purines andpyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil),dihydrouracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, .beta.-D-mannosyl-queosine, wybutoxosine, andphosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine, pseudouridine,5-methylcytidine and inosine. The preparation of such analogues is knownto a person skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071,4,401,796, 4,415,732, 4,458,066. U.S. Pat. Nos. 4,500,707, 4,668,777,4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, thedisclosure of which is included here in its full scope by reference.

Typically, mRNA synthesis includes the addition of a “cap” on the 5′end, and a “tail” on the 3′ end. The presence of the cap is important inproviding resistance to nucleases found in most eukaryotic cells. Thepresence of a “tail” serves to protect the mRNA from exonucleasedegradation.

Thus, in some embodiments, mRNAs include a 5′ cap structure. A 5′ cap istypically added as follows: first, an RNA terminal phosphatase removesone of the terminal phosphate groups from the 5′ nucleotide, leaving twoterminal phosphates; guanosine triphosphate (GTP) is then added to theterminal phosphates via a guanylyl transferase, producing a 5′5′5triphosphate linkage; and the 7-nitrogen of guanine is then methylatedby a methyltransferase. 2′-O-methylation may also occur at the firstbase and/or second base following the 7-methyl guanosine triphosphateresidues. Examples of cap structures include, but are not limited to,m7GpppNp-RNA, m7GpppNmp-RNA and m7GpppNmpNmp-RNA (where m indicates2′-Omethyl residues).

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

While mRNA provided from in vitro transcription reactions may bedesirable in some embodiments, other sources of mRNA are contemplated aswithin the scope of the invention including mRNA produced from bacteria,fungi, plants, and/or animals.

The present invention may be used to formulate and encapsulate mRNAsencoding a variety of proteins. Non-limiting examples of mRNAs suitablefor the present invention include mRNAs encoding spinal motor neuron 1(SMN), alpha-galactosidase (GLA), argininosuccinate synthetase (ASS1),ornithine transcarbamylase (OTC), Factor IX (FIX), phenylalaninehydroxylase (PAH), erythropoietin (EPO), cystic fibrosis transmembraneconductance receptor (CFTR) and firefly luciferase (FFL). Exemplary mRNAsequences as disclosed herein are listed below:

Codon-Optimized Henman OTC Coding Sequence (SEQ ID NO: 1)AUGCUGUUCAACCUUCGGAUCUUGCUGAACAACGC UGCGUUCCGGAAUGGUCACAACUUCAUGGUCCGGAACUUCAGAUGCGGCCAGCCGCUCCAGAACAAGGUG CAGCUCAAGGGGAGGGACCUCCUCACCCUGAAAAACUUCACCGGAGAAGAGAUCAAGUACAUGCUGUGGC UGUCAGCCGACCUCAAAUUCCGGAUCAAGCAGAAGGGCGAAUACCUUCCUUUGCUGCAGGGAAAGUCCCU GGGGAUGAUCUUCGAGAAGCGCAGCACUCGCACUAGACUGUCAACUGAAACCGGCUUCGCGCUGCUGOGA GGACACCCCUGCUUCCUGACCACCCAAGAUAUCCAUCUGGGUGUGAACGAAUCCCUCACCGACACAGCGC GGGUGCUGUCGUCCAUGGCAGACGCGGUCCUCGCCCGCGUGUACAAGCAGUCUGAUCUGGACACUCUGGC CAAGGAAGCCUCCAUUCCUAUCAUUAAUGGAUUGUCCGACCUCUACCAUCCCAUCCAGAUUCUGGCCGAU UAUCUGACUCUGCAAGAACAUUACAGCUCCCUGAAGGGGCUUACCCUUUCGUGGAUCGGCGACGGCAACA ACAUUCUGCACAGCAUUAUGAUGAGCGCUGCCAAGUUUGGAAUGCACCUCCAAGCAGCGACCCCGAAGGG AUACGAGCCAGACGCCUCCGUGACGAAGCUGGCUGAGCAGUACGCCAAGGAGAACGGCACUAAGCUGCUG CUCACCAACGACCCUCUCGAAGCCGCCCACGGUGGCAACGUGCUGAUCACCGAUACCUGGAUCUCCAUGG GACAGGAGGAGGAAAAGAAGAAGCGCCUGCAAGCAUUUCAGGGGUACCAGGUGACUAUGAAAACCGCCAA GGUCGCCGCCUCGGACUGGACCUUCUUGCACUGUCUGCCCAGAAAGCCCGAAGAGGUGGACGACGAGGUG UUCUACAGCCCGCGGUCGCUGGUCUUUCCGGAGGCCGAAAACAGGAAGUGGACUAUCAUGGCCGUGAUGG UGUCCCUGCUGACCGAUUACUCCCCGCAGCUGCAGAAACCAAAGUUCUGA Codon-Optimized Human ASS1 Coding Sequence(SEQ ID NO: 2) AUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACCAGCUGCAUCCUGGUGUGGC UGAAGGAGCAGGGCUACGACGUGAUCGCCUACCUGGCCAACAUCGGCCAGAAGGAGGACUUCGAGGAGGC CCGCAAGAAGGCCCUGAAGCUGGGCGCCAAGAAGGUGUUCAUCGAGGACGUGAGCCGCGAGUUCGUGGAG GAGUUCAUCUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGACCGCUACCUGCUGGGCACCAGCCUGG CCCGCCCCUGCAUCGCCCGCAAGCAGGUGGAGAUCGCCCAGCOCGAGGGCGCCAAGUACGUGAGCCACGG CGCCACCGGCAAGGGCAACGACCAGGUGCGCUUCGAGCUGAGCUGCUACAGCCUGGCCCCCCAGAUCAAG GUGAUCGCCCCCUGGCGCAUGCCCGAGUUCUACAACCGCUUCAAGGGCCGCAACGACCUGAUGGAGUACG CCAAGCAGCACGGCAUCCCCAUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGAGAACCUGAUGCA CAUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAACCAGGCCCCCCCCGGCCUGUACACCAAGACC CAGGACCCCGCCAAGGCCCCCAACACCCCCGACAUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUGA AGGUGACCAACGUGAAGGACGGCACCACCCACCAGACCAGCCUGGAGCUGUUCAUGUACCUGAACGAGGU GGCCGGCAAGCACGGCGUGGGCCGCAUCGACAUCGUGGAGAACCGCUUCAUCGGCAUGAAGAGCCGCGGC AUCUACGAGACCCCCGCCGGCACCAUCCUGUACCACGCCCACCUGGACAUCGAGGCCUUCACCAUGGACC GCGAGGUGCGCAAGAUCAAGCAGGGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACCGGCUUCUGGCA CAGCCCCGAGUGCGAGUUCGUGCGCCACUGCAUCGCCAAGAGCCAGGAGCGCGUGGAGGGCAAGGUGCAG GUGAGCGUGCUGAAGGGCCAGGUGUACAUCCUGGGCCGCGAGAGCCCCCUGAGCCUGUACAACGAGGAGC UGGUGAGCAUGAACGUGCAGGGCGACUACGAGCCCACCGACGCCACCGGCUUCAUCAACAUCAACAGCCU GCGCCUGAAGGAGUACCACCGCCUGCAGAGCAAGGUGACCGCCAAGUGA Codon-Optimized Human CFTR Coding Sequence (SEQ ID NO: 3)AUGCAACGCUCUCCUCUUGAAAAGGCCUCGGUGGU GUCCAAGCUCUUCUUCUCGUGGACUAGACCCAUCCUGAGAAAGGGGUACAGACAGCGCUUGGAGCUGUCC GAUAUCUAUCAAAUCCCUUCCGUGGACUCCGCGGACAACCUGUCCGAGAAGCUCGAGAGAGAAUGGGACA GAGAACUCGCCUCAAAGAAGAACCCGAAGCUGAUUAAUGCGCUUAGGCGGUGCUUUUUCUGGCGGUUCAU GUUCUACGGCAUCUUCCUCUACCUGGGAGAGGUCACCAAGGCCGUGCAGCCCCUGUUGCUGGGACGGAUU AUUGCCUCCUACGACCCCGACAACAAGGAAGAAAGAAGCAUCGCUAUCUACUUGGGCAUCGGUCUGUGCC UGCUUUUCAUCGUCCGGACCCUCUUGUUGCAUCCUGCUAUUUUCGGCCUGCAUCACAUUGGCAUGCAGAU GAGAAUUGCCAUGUUUUCCCUGAUCUACAAGAAAACUCUGAAGCUCUCGAGCCGCGUGCUUGACAAGAUU UCCAUCGGCCAGCUCGUGUCCCUGCUCUCCAACAAUCUGAACAAGUUCGACGAGGGCCUCGCCCUGGCCC ACUUCGUGUGGAUCGCCCCUCUGCAAGUGGCGCUUCUGAUGGGCCUGAUCUGGGAGCUGCUGCAAGCCUC GGCAUUCUGUGGGCUUGGAUUCCUGAUCGUGCUGGCACUGUUCCAGGCCGGACUGGGGCGGAUGAUGAUG AAGUACAGGGACCAGAGAGCCGGAAAGAUUJUCCGAACGGCUGGUGAUCACUUCGGAAAUGAUCGAAAAC AUCCAGUCAGUGAAGGCCUACUGCUGGGAAGAGGCCAUGGAAAAGAUGAUUGAAAACCUCCGGCAAACCG AGCUGAAGCUGACCCGCAAGGCCGCUUACGUGCGCUAUUUCAACUCGUCCGCUUUCUUCUUCUCCGGGUU CUUCGUGGUGUUUCUCUCCGUGCUCCCCUACGCCCUGAUUAAGGGAAUCAUCCUCAGGAAGAUCUUCACC ACCAUUUCCUUCUGUAUCGUGCUCCGCAUGGCCGUGACCCGGCAGUUCCCAUGGGCCGUGCAGACUUGGU ACGACUCCCUGGGAGCCAUUAACAAGAUCCAGGACUUCCUUCAAAAGCAGGAGUACAAGACCCUCGAGUA CAACCUGACUACUACCGAGGUCGUGAUGGAAAACGUCACCGCCUUUUGGGAGGAGGGAUUUGGCGAACUG UUCGAGAAGGCCAAGCAGAACAACAACAACCGCAAGACCUCGAACGGUGACGACUCCCUCUUCUUUUCAA ACUUCAGCCUGCUCGGGACGCCCGUGCUGAAGGACAUUAACUUCAAGAUCGAAAGAGGACAGCUCCUGGC GGUGGCCGGAUCGACCGGAGCCGGAAAGACUUCCCUGCUGAUGGUGAUCAUGGGAGAGCUUGAACCUAGC GAGGGAAAGAUCAAGCACUCCGGCCGCAUCAGCUUCUGUAGCCAGUUUUCCUGGAUCAUGCCCGGAACCA UUAAGGAAAACAUCAUCUUCGGCGUGUCCUACGAUGAAUACCGCUACCGGUCCGUGAUCAAAGCCUGCCA GCUGGAAGAGGAUAUUUCAAAGUUCGCGGAGAAAGAUAACAUCGUGCUGGGCGAAGGGGGUAUUACCUUG UCGGGGGGCCAGCGGGCUAGAAUCUCGCUGGCCAGAGCCGUGUAUAAGGACGCCGACCUGUAUCUCCUGG ACUCCCCCUUCGGAUACCUGGACGUCCUGACCGAAAAGGAGAUCUUCGAAUCGUGCGUGUGCAAGCUGAU GGCUAACAAGACUCGCAUCCUCGUGACCUCCAAAAUGGAGCACCUGAAGAAGGCAGACAAGAUUCUGAUU CUGCAUGAGGGGUCCUCCUACUUUUACGGCACCUUCUCGGAGUUGCAGAACUUGCAGCCCGACUUCUCAU CGAAGCUGAUGGGUUGCGACAGCUUCGACCAGUUCUCCGCCGAAAGAAGGAACUCGAUCCUGACGGAAAC CUUGCACCGCUUCUCUUUGGAAGGCGACGCCCCUGUGUCAUGGACCGAGACUAAGAAGCAGAGCUUCAAG CAGACCGGGGAAUUCGGCGAAAAGAGGAAGAACAGCAUCUUGAACCCCAUUAACUCCAUCCGCAAGUUCU CAAUCGUGCAAAAGACGCCACUGCAGAUGAACGGCAUUGAGGAGGACUCCGACGAACCCCUUGAGAGGCG CCUGUCCCUGGUGCCGGACAGCGAGCAGGGAGAAGCCAUCCUGCCUCGGAUUUCCGUGAUCUCCACUGGU CCGACGCUCCAAGCCCGGCGGCGGCAGUCCGUGCUGAACCUGAUGACCCACAGCGUGAACCAGGGCCAAA ACAUUCACCGCAAGACUACCGCAUCCACCCGGAAAGUGUCCCUGGCACCUCAAGCGAAUCUUACCGAGCU CGACAUCUACUCCCGGAGACUGUCGCAGGAAACCGGGCUCGAAAUUUCCGAAGAAAUCAACGAGGAGGAU CUGAAAGAGUGCUUCUUCGACGAUAUGGAGUCGAUACCCGCCGUGACGACUUGGAACACUUAUCUJGCGG UACAUCACUGUGCACAAGUCAUUGAUCUUCGUGCUGAUUUGGUGCCUGGUGAUUUUCCUGGCCGAGGUCG CGGCCUCACUGGUGGUGCUCUGGCUGUUGGGAAACACGCCUCUGCAAGACAAGGGAAACUCCACGCACUC GAGAAACAACAGCUAUGCCGUGAUUAUCACUUCCACCUCCUCUUAUUACGUGUUCUACAUCUACGUCGGA GUGGCGGAUACCCUGCUCGCGAUGGGUUUCUUCAGAGGACUGCCGCUGGUCCACACCUUGAUCACCGUCA GCAAGAUUCUUCACCACAAGAUGUUGCAUAGCGUGCUGCAGGCCCCCAUGUCCACCCUCAACACUCUGAA GGCCOGAGGCAUUCUGAACAGAUUCUCCAAGGACAUCGCUAUCCUGGACGAUCUCCUGCCGCUUACCAUC UUUGACUUCAUCCAGCUGCUGCUGAUCGUGAUUGGAGCAAUCGCAGUGGUGGCGGUGCUGCAGCCUUACA UUUUCGUGGCCACUGUGCCGGUCAUUGUGGCGUUCAUCAUGCUGCGGGCCUACUUCCUCCAAACCAGCCA GCAGCUGAAGCAACUGGAAUCCGAGGGACGAUCCCCCAUCUUCACUCACCUUGUGACGUCGUUGAAGGGA CUGUGGACCCUCCGGGCUUUCGGACGGCAGCCCUACUUCGAAACCCUCUUCCACAAGGCCCUGAACCUCC ACACCGCCAAUUGGUUCCUGUACCUGUCCACCCUGCGGUGGUUCCAGAUGCGCAUCGAGAUGAUUUUCGU CAUCUUCUUCAUCGCGGUCACAUUCAUCAGCAUCCUGACUACCGGAGAGGGAGAGGGACGGGUCGGAAUA AUCCUGACCCUCGCCAUGAACAUUAUGAGCACCCUGCAGUGGGCAGUGAACAGCUCGAUCGACGUGGACA GCCUGAUGCGAAGCGUCAGCCGCGUGUUCAAGUUCAUCGACAUGCCUACUGAGGGAAAACCCACUAAGUC CACUAAGCCCUACAAAAAUGGCCAGCUGAGCAAGGUCAUGAUCAUCGAAAACUCCCACGUGAAGAAGGAC GAUAUUUGGCCCUCCGGAGGUCAAAUGACCGUGAAGGACCUGACCGCAAAGUACACCGAGGGAGGAAACG CCAUUCUCGAAAACAUCAGCUUCUCCAUUUCGCCGGGACAGCGGGUCGGCCUUCUCGGGCGGACCGGUUC CGGGAAGUCAACUCUGCUGUCGGCUUUCCUCCGGCUGCUGAAUACCGAGGGGGAAAUCCAAAUUGACGGC GUGUCUUGGGAUUCCAUUACUCUGCAGCAGUGGCGGAAGGCCUUCGGCGUGAUCCCCCAGAAGGUGUUCA UCUUCUCGGGUACCUUCCGGAAGAACCUGGAUCCUUACGAGCAGUGGAGCGACCAAGAAAUCUGGAAGGU CGCCGACGAGGUCGGCCUGCGCUCCGUGAUUGAACAAUUUCCUGGAAAGCUGGACUUCGUGCUCGUCGAC GGGGGAUGUGUCCUGUCGCACGGACAUAAGCAGCUCAUGUGCCUCGCACGGUCCGUGCUCUCCAAGGCCA AGAUUCUGCUGCUGGACGAACCUUCGGCCCACCUGGAUCCGGUCACCUACCAGAUCAUCAGGAGGACCCU GAAGCAGGCCUUUGCCGAUUGCACCGUGAUUCUCUGCGAGCACCGCAUCGAGGCCAUGCUGGAGUGCCAG CAGUUCCUGGUCAUCGAGGAGAACAAGGUCCGCCAAUACGACUCCAUUCAAAAGCUCCUCAACGAGCGGU CGCUGUUCAGACAAGCUAUUUCACCGUCCGAUAGAGUGAAGCUCUUCCCGCAUCGGAACAGCUCAAAGUG CAAAUCGAAGCCGCAGAUCGCAGCCUUGAAGGAAGAGACUGAGGAAGAGGUGCAGGACACCCGGCUUUAAComparison Codon-Optimized Human CFTR mRNA Coding Sequence(SEQ ID NO: 4) AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCC UUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGA UAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUC AACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCA CAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACG GAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCA GCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGA CACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAA CCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUU UUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGG CAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGA AAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCU AUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGU AUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUU GAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUG ACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACU UCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGU GACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAG ACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACA UCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCU CUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUC UGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUG AGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGA UAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGA GCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAA AAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAU GGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUC UCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCA GCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGU AUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGU AUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAA UUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGC CAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUC AACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAG UGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGG ACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUC CCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCA UCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUAC GCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACA AGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAG GACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACU GCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUU GCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGG CUAUUGCAGUAGUCOCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAU CAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCU AUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACU UUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCG AUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUG ACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGC AGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAU CGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUA AUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGG ACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGG UCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUU CUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGA AAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUA UGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAG UUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCA UGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGA CCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGU GAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGU ACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGU GAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAG ACUGAAGAAGAAGUUCAAGACACGCGUCUUUAACodon-Optimized Human PAH Coding Sequence (SEQ ID NO: 5)AUGAGCACCGCCGUGCUGGAGAACCCCGGCCUGGG CCGCAAGCUGAGCGACUUCGGCCAGGAGACCAGCUACAUCGAGGACAACUGCAACCAGAACGGCGCCAUC AGCCUGAUCUUCAGCCUGAAGGAGGAGGUGGGCGCCCUGGCCAAGGUGCUGCGCCUGUUCGAGGAGAACG ACGUGAACCUGACCCACAUCGAGAGCCGCCCCAGCCGCCUGAAGAAGGACGAGUACGAGUUCUUCACCCA CCUGGACAAGCGCAGCCUGCCCGCCCUGACCAACAUCAUCAAGAUCCUGCGCCACGACAUCGGCGCCACC GUGCACGAGCUGAGCCGCGACAAGAAGAAGGACACCGUGCCCUGGUUCCCCCGCACCAUCCAGGAGCUGG ACCGCUUCGCCAACCAGAUCCUGAGCUACGGCGCCGAGCUGGACGCCGACCACCCCGGCUUCAAGGACCC CGUGUACCGCGCCCGCCGCAAGCAGUUCGCCGACAUCGCCUACAACUACCGCCACGGCCAGCCCAUCCCC CGCGUGGAGUACAUGGAGGAGGAGAAGAAGACCUGGGGCACCGUGUUCAAGACCCUGAAGAGCCUGUACA AGACCCACGCCUGCUACGAGUACAACCACAUCUUCCCCCUGCUGGAGAAGUACUGCGGCUUCCACGAGGA CAACAUCCCCCAGCUGGAGGACGUGAGCCAGUUCCUGCAGACCUGCACCGGCUUCCGCCUGCGCCCCGUG GCCGGCCUGCUGAGCAGCCGCGACUUCCUGGGCGGCCUGGCCUUCCGCGUGUUCCACUGCACCCAGUACA UCCGCCACGGCAGCAAGCCCAUGUACACCCCCGAGCCCGACAUCUGCCACGAGCUGCUGGGCCACGUGCC CCUGUUCAGCGACCGCAGCUUCGCCCAGUUCAGCCAGGAGAUCGGCCUGGCCAGCCUGGGCGCCCCCGAC GAGUACAUCGAGAAGCUGGCCACCAUCUACUGGUUCACCGUGGAGUUCGGCCUGUGCAAGCAGGGCGACA GCAUCAAGGCCUACGGCGCCGGCCUGCUGAGCAGCUUCGGCGAGCUGCAGUACUGCCUGAGCGAGAAGCC CAAGCUGCUGCCCCUGGAGCUGGAGAAGACCGCCAUCCAGAACUACACCGUGACCGAGUUCCAGCCCCUG UACUACGUGGCCGAGAGCUUCAACGACGCCAAGGAGAAGGUGCGCAACUUCGCCGCCACCAUCCCCCGCC CCUUCAGCGUGCGCUACGACCCCUACACCCAGCGCAUCGAGGUGCUGGACAACACCCAGCAGCUGAAGAU CCUGGCCGACAGCAUCAACAGCGAGAUCGGCAUCCUGUGCAGCGCCCUGCAGAAGAUCAAGUAA

In some embodiments, an mRNA suitable for the present invention has anucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical SEQID NO: 1. SEQ ID NO: 2, SEQ ID NO:3 or SEQ ID NO: 4. In someembodiments, an mRNA suitable for the present invention comprises anucleotide sequence identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3or SEQ ID NO: 4.

mRNA Solution

mRNA may be provided in a solution to be mixed with a lipid solutionsuch that the mRNA may be encapsulated in lipid nanoparticles. Asuitable mRNA solution may be any aqueous solution containing mRNA to beencapsulated at various concentrations below 1 mg/ml. For example, asuitable mRNA solution may contain an mRNA at a concentration of or lessthan about 0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, 0.04 mg/ml, 0.05 mg/ml,0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml,0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8mg/ml, 0.9 mg/ml, or 1.0 mg/ml.

Typically, a suitable mRNA solution may also contain a buffering agentand/or salt. Generally, buffering agents can include HEPES, ammoniumsulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassiumphosphate and sodium phosphate. In some embodiments, suitableconcentration of the buffering agent may range from about 0.1 mM to 100mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mMto 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or9 to 12 mM. In some embodiments, suitable concentration of the bufferingagent is or greater than about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.

Exemplary salts can include sodium chloride, magnesium chloride, andpotassium chloride. In some embodiments, suitable concentration of saltsin an mRNA solution may range from about 1 mM to 500 mM, 5 mM to 400 mM,10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mMto 150 mM, or 50 mM to 100 mM. Salt concentration in a suitable mRNAsolution is or greater than about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

In some embodiments, a suitable mRNA solution may have a pH ranging fromabout 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0,4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5. In some embodiments, asuitable mRNA solution may have a pH of or no greater than about 3.5,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.6,5.8, 6.0, 6.1, 6.3, and 6.5.

Various methods may be used to prepare an mRNA solution suitable for thepresent invention. In some embodiments, mRNA may be directly dissolvedin a buffer solution described herein. In some embodiments, an mRNAsolution may be generated by mixing an mRNA stock solution with a buffersolution prior to mixing with a lipid solution for encapsulation. Insome embodiments, an mRNA solution may be generated by mixing an mRNAstock solution with a buffer solution immediately before mixing with alipid solution for encapsulation. In some embodiments, a suitable mRNAstock solution may contain mRNA in water at a concentration at orgreater than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.8mg/ml, 1.0 mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, or 1.6 mg/ml, 2.0mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, or 5.0mg/ml.

In some embodiments, an mRNA stock solution is mixed with a buffersolution using a pump. Exemplary pumps include but are not limited togear pumps, peristaltic pumps and centrifugal pumps.

Typically, the buffer solution is mixed at a rate greater than that ofthe mRNA stock solution. For example, the buffer solution may be mixedat a rate at least 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, or 20×greater than the rate of the mRNA stock solution. In some embodiments, abuffer solution is mixed at a flow rate ranging between about 100-6000ml/minute (e.g., about 100-300 ml/minute, 300-600 ml/minute, 600-1200ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute). In someembodiments, a buffer solution is mixed at a flow rate of or greaterthan about 60 ml/minute, 100 ml/minute, 140 ml/minute, 180 ml/minute,220 mi/minute, 260 ml/minute, 300 ml/minute, 340 ml/minute, 380ml/minute, 420 mi/minute, 480 ml/minute, 540 ml/minute, 600 ml/minute,1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000m/minute.

In some embodiments, an mRNA stock solution is mixed at a flow rateranging between about 10-600 ml/minute (e.g., about 5-50 ml/minute,about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute,about 120-240 ml/minute, about 240-360 mi/minute, about 360-480mi/minute, or about 480-600 ml/minute). In some embodiments, an mRNAstock solution is mixed at a flow rate of or greater than about 5ml/minute, 10 ml/minute, 15 ml/minute, 20 ml/minute, 25 ml/minute, 30ml/minute, 35 ml/minute, 40 ml/minute, 45 ml/minute, 50 mi/minute, 60ml/minute, 80 ml/minute, 100 ml/minute, 200 ml/minute, 300 ml/minute,400 ml/minute, 500 ml/minute, or 600 ml/minute.

Lipid Solution

According to the present invention, a lipid solution contains a mixtureof lipids suitable to form lipid nanoparticles for encapsulation ofmRNA. In some embodiments, a suitable lipid solution is ethanol based.For example, a suitable lipid solution may contain a mixture of desiredlipids dissolved in pure ethanol (i.e., 100% ethanol). In anotherembodiment, a suitable lipid solution is isopropyl alcohol based. Inanother embodiment, a suitable lipid solution isdimethylsulfoxide-based. In another embodiment, a suitable lipidsolution is a mixture of suitable solvents including, but not limitedto, ethanol, isopropyl alcohol and dimethylsulfoxide.

A suitable lipid solution may contain a mixture of desired lipids atvarious concentrations. For example, a suitable lipid solution maycontain a mixture of desired lipids at a total concentration of about0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, 0.04 mg/ml, 0.05 mg/ml, 0.06 mg/ml,0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0mg/ml, 3.0 mg/ml, 4.0 mg/ml, 5.0 mg/ml, 6.0 mg/ml, 7.0 mg/ml, 8.0 mg/ml,9.0 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml,or 100 mg/ml. In some embodiments, a suitable lipid solution may containa mixture of desired lipids at a total concentration ranging from about0.1-100 mg/ml, 0.5-90 mg/ml, 1.0-80 mg/ml, 1.0-70 mg/ml, 1.0-60 mg/ml,1.0-50 mg/ml, 1.0-40 mg/ml, 1.0-30 mg/ml, 1.0-20 mg/ml, 1.0-15 mg/ml,1.0-10 mg/ml, 1.0-9 mg/ml, 1.0-8 mg/ml, 1.0-7 mg/ml, 1.0-6 mg/ml, or1.0-5 mg/ml. In some embodiments, a suitable lipid solution may containa mixture of desired lipids at a total concentration up to about 100mg/ml, 90 mg/ml, 80 mg/ml, 70 mg/ml, 60 mg/ml, 50 mg/ml, 40 mg/ml, 30mg/ml, 20 mg/ml, or 10 mg/ml.

Any desired lipids may be mixed at any ratios suitable for encapsulatingmRNAs. In some embodiments, a suitable lipid solution contains a mixtureof desired lipids including cationic lipids, helper lipids (e.g. noncationic lipids and/or cholesterol lipids) and/or PEGylated lipids. Insome embodiments, a suitable lipid solution contains a mixture ofdesired lipids including one or more cationic lipids, one or more helperlipids (e.g. non cationic lipids and/or cholesterol lipids) and one ormore PEGylated lipids.

Cationic Lipids

As used herein, the phrase “cationic lipids” refers to any of a numberof lipid species that have a net positive charge at a selected pH, suchas physiological pH.

Suitable cationic lipids for use in the compositions and methods of theinvention include the cationic lipids as described in InternationalPatent Publication WO 2010/144740, which is incorporated herein byreference. In certain embodiments, the compositions and methods of thepresent invention include a cationic lipid,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the present invention include ionizable cationic lipids as describedin International Patent Publication WO 2013/149140, which isincorporated herein by reference. In some embodiments, the compositionsand methods of the present invention include a cationic lipid of one ofthe following formulas:

or a pharmaceutically acceptable salt thereof, wherein R₁ and R₂ areeach independently selected from the group consisting of hydrogen, anoptionally substituted, variably saturated or unsaturated C₁-C₂₀ alkyland an optionally substituted, variably saturated or unsaturated C₆-C₂₀acyl; wherein L₁ and L₂ are each independently selected from the groupconsisting of hydrogen, an optionally substituted C₁-C₃₀ alkyl, anoptionally substituted variably unsaturated C₁-C₃₀ alkenyl, and anoptionally substituted C₁-C₃₀ alkynyl; wherein m and o are eachindependently selected from the group consisting of zero and anypositive integer (e.g., where m is three); and wherein n is zero or anypositive integer (e.g., where n is one). In certain embodiments, thecompositions and methods of the present invention include the cationiclipid (15Z, 18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (“HGT5000”), having a compound structureof:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include thecationic lipid (15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (“HGT5001”), having a compound structureof:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include thecationic lipid and(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (“HGT5002”), having a compound structureof:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include cationic lipids described as aminoalcohollipidoids in International Patent Publication WO 2010/053572, which isincorporated herein by reference. In certain embodiments, thecompositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2016/118725, which is incorporatedherein by reference. In certain embodiments, the compositions andmethods of the present invention include a cationic lipid having acompound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2016/118724, which is incorporatedherein by reference. In certain embodiments, the compositions andmethods of the present invention include a cationic lipid having acompound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include a cationic lipid having the formula of14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, andpharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publications WO 2013/063468 and WO 2016/205691,each of which are incorporated herein by reference. In some embodiments,the compositions and methods of the present invention include a cationiclipid of the following formula:

or pharmaceutically acceptable salts thereof, wherein each instance ofR^(L) is independently optionally substituted C₆-C₄₀ alkenyl. In certainembodiments, the compositions and methods of the present inventioninclude a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2015/184256, which is incorporatedherein by reference. In some embodiments, the compositions and methodsof the present invention include a cationic lipid of the followingformula:

or a pharmaceutically acceptable salt thereof, wherein each Xindependently is O or S; each Y independently is O or S; each mindependently is 0 to 20; each n independently is 1 to 6; each R_(A) isindependently hydrogen, optionally substituted C1-50 alkyl, optionallysubstituted C2-50 alkenyl, optionally substituted C2-50 alkynyl,optionally substituted C3-10 carbocyclyl, optionally substituted 3-14membered heterocyclyl, optionally substituted C6-14 aryl, optionallysubstituted 5-14 membered heteroaryl or halogen; and each RB isindependently hydrogen, optionally substituted C1-50 alkyl, optionallysubstituted C2-50 alkenyl, optionally substituted C2-50 alkynyl,optionally substituted C3-10 carbocyclyl, optionally substituted 3-14membered heterocyclyl, optionally substituted C6-14 aryl, optionallysubstituted 5-14 membered heteroaryl or halogen. In certain embodiments,the compositions and methods of the present invention include a cationiclipid, “Target 23”, having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2016/004202, which is incorporatedherein by reference. In some embodiments, the compositions and methodsof the present invention include a cationic lipid having the compoundstructure:

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

or a pharmaceutically acceptable salt thereof.

Other suitable cationic lipids for use in the compositions and methodsof the present invention include the cationic lipids as described in J.McClellan, M. C. King, Cell 2010, 141, 210-217 and in Whitehead et al.,Nature Communications (2014) 5:4277, which is incorporated herein byreference. In certain embodiments, the cationic lipids of thecompositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2015/199952, which is incorporatedherein by reference. In some embodiments, the compositions and methodsof the present invention include a cationic lipid having the compoundstructure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/004143, which is incorporatedherein by reference. In some embodiments, the compositions and methodsof the present invention include a cationic lipid having the compoundstructure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the resent invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the resent invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/075531, which is incorporatedherein by reference. In some embodiments, the compositions and methodsof the present invention include a cationic lipid of the followingformula:

or a pharmaceutically acceptable salt thereof, wherein one of L¹ or L²is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x), —S—S—, —C(═O)S—,—SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—,—OC(═O)NR^(a)—, or —NR^(a)C(═O)O—; and the other of L¹ or L² is—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x), —S—S—, —C(═O)S—, SC(═O)—,—NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or—NR^(a)C(═O)O— or a direct bond; G¹ and G² are each independentlyunsubstituted C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene; G³ is C₁-C₂aalkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈ cycloalkenylene;R^(a) is H or C₁-C₁₂ alkyl; R¹ and R² are each independently C₆-C₂₄alkyl or C₆-C₂₄ alkenyl; R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴; R⁴ is C₁-C₁₂ alkyl; R⁵ is H or C₁-C₆ alkyl; and x is 0, 1 or 2.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/117528, which is incorporatedherein by reference. In some embodiments, the compositions and methodsof the present invention include a cationic lipid having the compoundstructure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/049245, which is incorporatedherein by reference. In some embodiments, the cationic lipids of thecompositions and methods of the present invention include a compound ofone of the following formulas:

and pharmaceutically acceptable salts thereof. For any one of these fourformulas, R₄ is independently selected from —(CH₂)_(n)Q and—(CH₂)_(n)CHQR; Q is selected from the group consisting of —OR, —OH,—O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R), anda heterocycle; and n is 1, 2, or 3. In certain embodiments, thecompositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/173054 and WO 2015/095340, eachof which is incorporated herein by reference. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the present invention include cleavable cationic lipids as describedin International Patent Publication WO 2012/170889, which isincorporated herein by reference. In some embodiments, the compositionsand methods of the present invention include a cationic lipid of thefollowing formula:

wherein R₁ is selected from the group consisting of imidazole,guanidinium, amino, imine, enamine, an optionally-substituted alkylamino (e.g., an alkyl amino such as dimethylamino) and pyridyl; whereinR₂ is selected from the group consisting of one of the following twoformulas:

and wherein R₃ and R₄ are each independently selected from the groupconsisting of an optionally substituted, variably saturated orunsaturated C₆-C₂₀ alkyl and an optionally substituted, variablysaturated or unsaturated C₆-C₂₀ acyl; and wherein n is zero or anypositive integer (e.g., one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty or more). In certain embodiments,the compositions and methods of the present invention include a cationiclipid, “HGT4001”, having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid, “HGT4002”, having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid, “HGT4003”, having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid, “HGT4004”, having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid “HGT4005”, having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the present invention include cleavable cationic lipids as describedin U.S. Provisional Application No. 62/672,194, filed May 16, 2018, andincorporated herein by reference. In certain embodiments, thecompositions and methods of the present invention include a cationiclipid that is any of general formulas or any of structures (1a)-(21a)and (1b)-(21b) and (22)-(237) described in U.S. Provisional ApplicationNo. 62/672,194. In certain embodiments, the compositions and methods ofthe present invention include a cationic lipid that has a structureaccording to Formula (I′),

wherein:

-   -   R^(X) is independently —H, -L¹-R¹, or -L^(5A)-L^(5B)-B′;    -   each of L¹, L², and L³ is independently a covalent bond, —C(O)—,        —C(O)O—, —C(O)S—, or —C(O)NR^(L)—;    -   each L^(4A) and L^(5A) is independently —C(O)—, —C(O)O—, or        —C(O)NR^(L)—;    -   each L^(4B) and L^(5B) is independently C₁-C₂₀ alkylene; C₂-C₂₀        alkenylene; or C₂-C₂₀ alkynylene;    -   each B and B′ is NR⁴R⁵ or a 5- to 10-membered        nitrogen-containing heteroaryl;    -   each R¹, R², and R³ is independently C₆-C₃₀ alkyl, C₆-C₃₀        alkenyl, or C₆-C₃₀ alkynyl;    -   each R⁴ and R⁵ is independently hydrogen, C₁-C₁₀ alkyl; C₂-C₁₀        alkenyl; or C₂-C₁₀ alkynyl; and    -   each R^(L) is independently hydrogen, C₁-C₂₀ alkyl, C₂-C₂₀        alkenyl, or C₂-C₂₀ alkynyl.        In certain embodiments, the compositions and methods of the        present invention include a cationic lipid that is        Compound (139) of 62/672,194, having a compound structure of:

In some embodiments, the compositions and methods of the presentinvention include the cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (“DOTMA”).(Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No.4,897,355, which is incorporated herein by reference). Other cationiclipids suitable for the compositions and methods of the presentinvention include, for example, 5-carboxyspermylglycinedioctadecylamide(“DOGS”);2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium(“DOSPA”) (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989), U.S.Pat. Nos. 5,171,678; 5,334,761); 1,2-Dioleoyl-3-Dimethylammonium-Propane(“DODAP”); 1,2-Dioleoyl-3-Trimethylammonium-Propane (“DOTAP”).

Additional exemplary cationic lipids suitable for the compositions andmethods of the present invention also include:1,2-distearyloxy-N,N-dimethyl-3-aminopropane (“DSDMA”);1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (“DODMA”);1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (“DLinDMA”);1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (“DLenDMA”);N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”);3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(“CLinDMA”); 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy1-1-(cis,cis-9′, 1-2′-octadecadienoxy)propane (“CpLinDMA”);N,N-dimethyl-3,4-dioleyloxybenzylamine (“DMOBA”);1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (“DOcarbDAP”);2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (“DLinDAP”);1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (“DLincarbDAP”);1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (“DLinCDAP”);2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (“DLin-K-DMA”);2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9, 12-dien-1-yloxy]propane-1-amine (“Octyl-CLinDMA”);(2R)-2-((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-1-yloxy]propan-1-amine(“Octyl-CLinDMA (2R)”);(2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, fsl-dimethyl 3-[(9Z,12Z)-octadeca-9, 12-dien-1-yloxy]propan-1-amine (“Octyl-CLinDMA (2S)”);2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (“DLin-K-XTC2-DMA”);and2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(“DLin-KC2-DMA”) (see, WO 2010/042877, which is incorporated herein byreference; Semple et al., Nature Biotech. 28: 172-176 (2010)). (Heyes,J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V.,et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); International PatentPublication WO 2005/121348). In some embodiments, one or more of thecationic lipids comprise at least one of an imidazole, dialkylamino, orguanidinium moiety.

In some embodiments, one or more cationic lipids suitable for thecompositions and methods of the present invention include 2,2-Dilinoley1-4-dimethylaminoethyl-[1,3]-dioxolane (“XTC”);(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(“ALNY-100”) and/or4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide(“NC98-5”).

In some embodiments, the compositions of the present invention includeone or more cationic lipids that constitute at least about 5%, 10%, 20%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured by weight, ofthe total lipid content in the composition, e.g., a lipid nanoparticle.In some embodiments, the compositions of the present invention includeone or more cationic lipids that constitute at least about 5%, 10%, 20%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured as a mol %, ofthe total lipid content in the composition, e.g., a lipid nanoparticle.In some embodiments, the compositions of the present invention includeone or more cationic lipids that constitute about 30-70% (e.g., about30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about30-40%, about 35-50%, about 35-45%, or about 35-40%), measured byweight, of the total lipid content in the composition, e.g., a lipidnanoparticle. In some embodiments, the compositions of the presentinvention include one or more cationic lipids that constitute about30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%,about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about35-40%), measured as mol %, of the total lipid content in thecomposition, e.g., a lipid nanoparticle

In some embodiments, sterol-based cationic lipids may be use instead orin addition to cationic lipids described herein. Suitable sterol-basedcationic lipids are dialkylamino-, imidazole-, andguanidinium-containing sterol-based cationic lipids. For example,certain embodiments are directed to a composition comprising one or moresterol-based cationic lipids comprising an imidazole, for example, theimidazole cholesterol ester or “ICE” lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by structure (I) below.In certain embodiments, a lipid nanoparticle for delivery of RNA (e.g.,mRNA) encoding a functional protein may comprise one or moreimidazole-based cationic lipids, for example, the imidazole cholesterolester or “ICE” lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by the followingstructure:

In some embodiments, the percentage of cationic lipid in a liposome maybe greater than 10%, greater than 20%, greater than 30%, greater than40%, greater than 50%, greater than 60%, or greater than 70%. In someembodiments, cationic lipid(s) constitute(s) about 30-50% (e.g., about30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) ofthe liposome by weight. In some embodiments, the cationic lipid (e.g.,ICE lipid) constitutes about 30%, about 35%, about 40%, about 45%, orabout 50% of the liposome by molar ratio.

As used herein, the phrase “non-cationic lipid” refers to any neutral,zwitterionic or anionic lipid. As used herein, the phrase “anioniclipid” refers to any of a number of lipid species that carry a netnegative charge at a selected H, such as physiological pH. Non-cationiclipids include, but are not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine,sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixturethereof.

In some embodiments, such non-cationic lipids may be used alone, but arepreferably used in combination with other lipids, for example, cationiclipids. In some embodiments, the non-cationic lipid may comprise a molarratio of about 5% to about 90%, or about 10% to about 70% of the totallipid present in a liposome. In some embodiments, a non-cationic lipidis a neutral lipid, i.e., a lipid that does not carry a net charge inthe conditions under which the composition is formulated and/oradministered. In some embodiments, the percentage of non-cationic lipidin a liposome may be greater than 5%, greater than 10%, greater than20%, greater than 30%, or greater than 40%.

Suitable cholesterol-based cationic lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335), or ICE. In some embodiments, thecholesterol-based lipid may comprise a molar ration of about 2% to about30%, or about 5% to about 20% of the total lipid present in a liposome.In some embodiments, the percentage of cholesterol-based lipid in thelipid nanoparticle may be greater than 5%, greater than 10%, greaterthan 20%, greater than 30%, or greater than 40%.

The use of polyethylene glycol (PEG)-modified phospholipids andderivatized lipids such as derivatized ceramides (PEG-CER), includingN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000](C8 PEG-2000 ceramide) is also contemplated by the present invention,either alone or preferably in combination with other lipid formulationstogether which comprise the transfer vehicle (e.g., a lipidnanoparticle). Contemplated PEG-modified lipids include, but are notlimited to, a polyethylene glycol chain of up to S kDa in lengthcovalently attached to a lipid with alkyl chain(s) of C₆-C₂₀ length. Theaddition of such components may prevent complex aggregation and may alsoprovide a means for increasing circulation lifetime and increasing thedelivery of the lipid-nucleic acid composition to the target tissues,(Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may beselected to rapidly exchange out of the formulation in vivo (see U.S.Pat. No. 5,885,613). Particularly useful exchangeable lipids arePEG-ceramides having shorter acyl chains (e.g., C14 or C18). ThePEG-modified phospholipid and derivatized lipids of the presentinvention may comprise a molar ratio from about 0% to about 20%, about0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, orabout 2% of the total lipid present in the liposomal transfer vehicle.

According to various embodiments, the selection of cationic lipids,non-cationic lipids and/or PEG-modified lipids which comprise the lipidnanoparticle, as well as the relative molar ratio of such lipids to eachother, is based upon the characteristics of the selected lipid(s), thenature of the intended target cells, the characteristics of the mRNA tobe delivered. Additional considerations include, for example, thesaturation of the alkyl chain, as well as the size, charge, pH, pKa,fusogenicity and toxicity of the selected lipid(s). Thus the molarratios may be adjusted accordingly.

In some embodiments, a suitable delivery vehicle is formulated using apolymer as a carrier, alone or in combination with other carriersincluding various lipids described herein. Thus, in some embodiments,liposomal delivery vehicles, as used herein, also encompassnanoparticles comprising polymers. Suitable polymers may include, forexample, polyacrylates, polyalkycyanoacrylates, polylactide,polylactide-polyglycolide copolymers, polycaprolactones, dextran,albumin, gelatin, alginate, collagen, chitosan, cyclodextrins,protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine(PEI). When PEI is present, it may be branched PEI of a molecular weightranging from 10 to 40 kDa, e.g., 25 kDa branched PEI (Sigma #408727).

A suitable liposome for the present invention may include one or more ofany of the cationic lipids, non-cationic lipids, cholesterol lipids,PEG-modified lipids and/or polymers described herein at various ratios.As non-limiting examples, a suitable liposome formulation may include acombination selected from cKK-E12, DOPE, cholesterol and DMG-PEG2K;C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol andDMG-PEG2K: ICE, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, andDMG-PEG2K.

In various embodiments, cationic lipids (e.g., cKK-E12, C12-200, ICE,and/or HGT4003) constitute about 30-60% (e.g., about 30-55%, about30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about35-40%) of the liposome by molar ratio. In some embodiments, thepercentage of cationic lipids (e.g., cKK-E12, C12-200, ICE, and/orHGT4003) is or greater than about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, or about 60% of the liposome by molar ratio.

In some embodiments, the ratio of cationic lipid(s) to non-cationiclipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) may bebetween about 30-60:25-35:20-30:1-15, respectively. In some embodiments,the ratio of cationic lipid(s) to non-cationic lipid(s) tocholesterol-based lipid(s) to PEG-modified lipid(s) is approximately40:30:20:10, respectively. In some embodiments, the ratio of cationiclipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) toPEG-modified lipid(s) is approximately 40:30:25:5, respectively. In someembodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) tocholesterol-based lipid(s) to PEG-modified lipid(s) is approximately40:32:25:3, respectively. In some embodiments, the ratio of cationiclipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) toPEG-modified lipid(s) is approximately 50:25:20:5. In some embodiments,the ratio of sterol lipid(s) to non-cationic lipid(s) to PEG-modifiedlipid(s) is 50:45:5. In some embodiments, the ratio of sterol lipid(s)to non-cationic lipid(s) to PEG-modified lipid(s) is 50:40:10. In someembodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) toPEG-modified lipid(s) is 55:40:5. In some embodiments, the ratio ofsterol lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is55:35:10. In some embodiments, the ratio of sterol lipid(s) tonon-cationic lipid(s) to PEG-modified lipid(s) is 60:35:5. In someembodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) toPEG-modified lipid(s) is 60:30:10.

In some embodiments, a suitable liposome for the present inventioncomprises ICE and DOPE at an ICE:DOPE molar ratio of >1:1. In someembodiments, the ICE:DOPE molar ratio is <2.5:1. In some embodiments,the ICE:DOPE molar ratio is between 1:1 and 2.5:1. In some embodiments,the ICE:DOPE molar ratio is approximately 1.5:1. In some embodiments,the ICE:DOPE molar ratio is approximately 1.7:1. In some embodiments,the ICE:DOPE molar ratio is approximately 2:1. In some embodiments, asuitable liposome for the present invention comprises ICE and DMG-PEG-2Kat an ICE:DMG-PEG-2K molar ratio of >10:1. In some embodiments, theICE:DMG-PEG-2K molar ratio is <16:1. In some embodiments, theICE:DMG-PEG-2K molar ratio is approximately 12:1. In some embodiments,the ICE:DMG-PEG-2K molar ratio is approximately 14:1. In someembodiments, a suitable liposome for the present invention comprisesDOPE and DMG-PEG-2K at a DOPE: DMG-PEG-2K molar ratio of >5:1. In someembodiments, the DOPE: DMG-PEG-2K molar ratio is <11:1. In someembodiments, the DOPE: DMG-PEG-2K molar ratio is approximately 7:1. Insome embodiments, the DOPE: DMG-PEG-2K molar ratio is approximately10:1. In some embodiments, a suitable liposome for the present inventioncomprises ICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar ratioof 50:45:5. In some embodiments, a suitable liposome for the presentinvention comprises ICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2Kmolar ratio of 50:40:10. In some embodiments, a suitable liposome forthe present invention comprises ICE, DOPE and DMG-PEG-2K at anICE:DOPE:DMG-PEG-2K molar ratio of 55:40:5. In some embodiments, asuitable liposome for the present invention comprises ICE, DOPE andDMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar ratio of 55:35:10. In someembodiments, a suitable liposome for the present invention comprisesICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar ratio of60:35:5. In some embodiments, a suitable liposome for the presentinvention comprises ICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2Kmolar ratio of 60:30:10.

PEGylated Lipids

In some embodiments, a suitable lipid solution includes one or morePEGylated lipids. For example, the use of polyethylene glycol(PEG)-modified phospholipids and derivatized lipids such as derivatizedceramides (PEG-CER), includingN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000](C8 PEG-2000 ceramide) is also contemplated by the present invention.Contemplated PEG-modified lipids include, but are not limited to, apolyethylene glycol chain of up to 2 kDa, up to 3 kDa, up to 4 kDa or upto 5 kDa in length covalently attached to a lipid with alkyl chain(s) ofC₆-C₂₀ length. In some embodiments, a PEG-modified or PEGylated lipid isPEGylated cholesterol or PEG-2K. In some embodiments, particularlyuseful exchangeable lipids are PEG-ceramides having shorter acyl chains(e.g., C₁₄ or C₁₈).

PEG-modified phospholipid and derivatized lipids may constitute nogreater than about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% ofthe total lipids in a suitable lipid solution by weight or by molar. Insome embodiments, PEG-modified lipids may constitute about 5% or less ofthe total lipids in a suitable lipid solution by weight or by molarconcentration. In some embodiments, PEG-modified lipids may constituteabout 4% or less of the total lipids in a suitable lipid solution byweight or by molar concentration. In some embodiments, PEG-modifiedlipids typically constitute 3% or less of total lipids in a suitablelipid solution by weight or by molar concentration. In some embodiments,PEG-modified lipids typically constitute 2% or less of total lipids in asuitable lipid solution by weight or by molar concentration. In someembodiments, PEG-modified lipids typically constitute 1% or less oftotal lipids in a suitable lipid solution by weight or by molarconcentration. In some embodiments. PEG-modified lipids constitute about1-5%, about 1-4%, about 1-3%, or about 1-2%, of the total lipids in asuitable lipid solution by weight or by molar concentration. In someembodiments, PEG modified lipids constitute about 0.01-3% (e.g., about0.01-2.5%, 0.01-2%, 0.01-1.5%, 0.01-1%) of the total lipids in asuitable lipid solution by weight or by molar concentration.

Various combinations of lipids, i.e., cationic lipids, non-cationiclipids, PEG-modified lipids and optionally cholesterol, that can used toprepare, and that are comprised in, pre-formed lipid nanoparticles aredescribed in the literature and herein. For example, a suitable lipidsolution may contain cKK-E12. DOPE, cholesterol, and DMG-PEG2K; C12-200,DOPE, cholesterol, and DMG-PEG2K; HGT5000, DOPE, cholesterol, andDMG-PEG2K; HGT5001, DOPE, cholesterol, and DMG-PEG2K; cKK-E12, DPPC,cholesterol, and DMG-PEG2K; C12-200, DPPC, cholesterol, and DMG-PEG2K:HGT5000, DPPC, chol, and DMG-PEG2K; HGT5001, DPPC, cholesterol, andDMG-PEG2K; or ICE, DOPE and DMG-PEG2K. Additional combinations of lipidsare described in the art, e.g., U.S. Ser. No. 62/420,421 (filed on Nov.10, 2016), U.S. Ser. No. 62/421,021 (filed on Nov. 11, 2016), U.S. Ser.No. 62/464,327 (filed on Feb. 27, 2017), and PCT Application entitled“Novel ICE-based Lipid Nanoparticle Formulation for Delivery of mRNA,”filed on Nov. 10, 2017, the disclosures of which are included here intheir full scope by reference. The selection of cationic lipids,non-cationic lipids and/or PEG-modified lipids which comprise the lipidmixture as well as the relative molar ratio of such lipids to eachother, is based upon the characteristics of the selected lipid(s) andthe nature of the and the characteristics of the mRNA to beencapsulated. Additional considerations include, for example, thesaturation of the alkyl chain, as well as the size, charge, pH, pKa,fusogenicity and toxicity of the selected lipid(s). Thus the molarratios may be adjusted accordingly.

Pre-Formed Nanoparticle Formation and Mixing Process

The present invention is based on the surprising discovery that mixingempty pre-formed lipid nanoparticles (i.e., lipid nanoparticles formedin the absence of mRNA) and mRNA at a low concentration can result inefficient encapsulation without aggregation of lipid nanoparticles. Thisinvention is particularly useful in encapsulating mRNA with pre-formedlipid nanoparticles containing low levels of PEG-modified lipids (e.g.,no greater than 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the totallipids in the LNP). Without wishing to be bound by any theory, it isbelieved that lipid nanoparticles containing low levels of PEG-modifiedlipids tend to aggregate.

In some previously disclosed processes, see U.S. patent application Ser.No. 14/790,562 entitled “Encapsulation of messenger RNA”, filed Jul. 2,2015 and its provisional U.S. patent application Ser. No. 62/020,163,filed Jul. 2, 2014, the disclosure of which are hereby incorporated intheir entirety, in some embodiments, the previous invention provides aprocess of encapsulating messenger RNA (mRNA) in lipid nanoparticles bymixing an mRNA solution and a lipid solution, wherein the mRNA solutionand/or the lipid solution are heated to a pre-determined temperaturegreater than ambient temperature prior to mixing, to form lipidnanoparticles that encapsulate mRNA.

The present invention relates to a novel process for preparing a lipidnanoparticle containing mRNA, which involves combining pre-formed lipidnanoparticles with mRNA, wherein the pre-formed lipid nanoparticlescomprise low PEG-modified lipids (typically 3% or less of the totallipids in the LNP). In some embodiments, LNP concentrations can belowered (diluted) to 1 mg/ml, with simultaneous lowering (diluting) ofmRNA concentration to about 1 mg/ml to avoid LNP aggregation and ensurehigh efficiency of encapsulation. In some embodiments, the LNPconcentration is lowered to about 0.9 mg/ml or less, or 0.8 mg/ml orless, or 0.7 mg/ml or less, or 0.6 mg/ml or less, or 0.5 mg/ml or less,or 0.4 mg/ml or less, or 0.3 mg/ml or less, or 0.2 mg/ml or less, or 0.1mg/ml or less, or 0.05 mg/ml or less, or 0.01 mg/ml. In someembodiments, the corresponding mRNA concentration is lowered to about 3mg/ml or less, or 2 mg/ml or less, or 1 mg/ml or less, or 0.9 mg/ml orless, or 0.8 mg/ml or less, or 0.7 mg/ml or less, or 0.6 mg/ml or less,or 0.5 mg/ml or less, or 0.4 mg/ml or less, or 0.3 mg/ml or less, or 0.2mg/ml or less, or 0.1 mg/ml or less, or 0.05 mg/ml or less, or 0.01mg/ml. In some embodiments, LNP concentration in the encapsulationmixture is between 0.05 mg/ml and 2 mg/ml and the corresponding the mRNAconcentration is between 0.05 mg/ml and 2 mg/ml, such that the LNPparticles do not aggregate. In some embodiments, exemplary LNPconcentrations in the encapsulation mixture range between 0.1 mg/ml to 1mg/ml. In some embodiments, exemplary mRNA concentrations in the mRNAmixture range between 0.1 mg/ml to 1 mg/ml. In some embodiments, theconcentration of each of the pre-formed lipid nanoparticles and the mRNAis less than 1 mg/ml during mixing for encapsulation. The resultantformulated particles have high potency and efficacy. The mixing of thecomponents is achieved with pump systems which maintain the lipid/mRNA(N/P) ratio constant throughout the process and which also afford facilescale-up. In some embodiments, the process is performed at large scale.For example, in some embodiments, a composition according to the presentinvention contains at least about 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500mg, or 1000 mg of encapsulated mRNA.

For certain cationic lipid nanoparticle formulations of mRNA, in orderto achieve high encapsulation of mRNA, which is essential for protectionand delivery of mRNA, the mRNA in citrate buffer has to be heated. Inthose processes or methods, the heating is required to occur before theformulation process (i.e. heating the separate components) as heatingpost-formulation (post-formation of nanoparticles) does not increase theencapsulation efficiency of the mRNA in the lipid nanoparticles. Incontrast, in some embodiments of the novel processes of the presentinvention, the order of heating of mRNA does not appear to affect themRNA encapsulation percentage. In some embodiments, no heating (i.e.,maintaining at ambient temperature) of one or more of the solutioncomprising the pre-formed lipid nanoparticles, the solution comprisingthe mRNA and the mixed solution comprising the lipid nanoparticleencapsulated mRNA is required to occur before or after the formulationprocess. This potentially provides a huge advantage for preciselyscaling up, as controlled temperature change post-mixing is easier toachieve.

With this novel process, in some embodiments, encapsulating mRNA byusing a step of mixing the mRNA with empty (i.e., empty of mRNA)pre-formed lipid nanoparticles (Process B) results in remarkably higherpotency as compared to encapsulating mRNA by mixing the mRNA with justthe lipid components (i.e., that are not pre-formed into lipidnanoparticles)(Process A). As described in the Examples below, forexample in Tables 3 and 4, the potency of any mRNA encapsulated lipidnanoparticles tested is from more than 100% to more than 1000% morepotent when prepared by Process B as compared to Process A.

In some embodiments, the empty (i.e., empty of mRNA) lipid nanoparticleswithout mRNA are formed by mixing a lipid solution containing dissolvedlipids in a solvent, and an aqueous/buffer solution. In someembodiments, the solvent can be ethanol. In some embodiments, theaqueous solution can be a citrate buffer.

As used herein, the term “ambient temperature” refers to the temperaturein a room, or the temperature which surrounds an object of interest(e.g., a pre-formed empty lipid nanoparticle solution, an mRNA solution,or a lipid nanoparticle solution containing mRNA) without heating orcooling. In some embodiments, the ambient temperature at which one ormore of the solutions is maintained is or is less than about 35° C., 30°C., 25° C., 20° C., or 16° C. In some embodiments, the ambienttemperature at which one or more of the solutions is maintained rangesfrom about 15-35° C., about 15-30° C., about 15-25° C., about 15-20° C.,about 20-35° C., about 25-35° C. about 30-35° C. about 20-30° C., about25-30° C. or about 20-25° C. In some embodiments, the ambienttemperature at which one or more of the solutions is maintained is20-25° C.

Therefore, a pre-determined temperature greater than ambient temperatureis typically greater than about 25° C. In some embodiments, apre-determined temperature suitable for the present invention is or isgreater than about 30° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60°C., 65° C., or 70° C. In some embodiments, a pre-determined temperaturesuitable for the present invention ranges from about 25-70° C., about30-70° C., about 35-70° C., about 40-70° C., about 45-70° C. about50-70° C., or about 60-70° C. In particular embodiments, apre-determined temperature suitable for the present invention is about65° C.

In some embodiments, the mRNA, or pre-formed empty (i.e., empty of mRNA)lipid nanoparticle solution, or both, may be heated to a pre-determinedtemperature above the ambient temperature prior to mixing. In someembodiments, the mRNA and the pre-formed empty lipid nanoparticlesolution are heated to the pre-determined temperature separately priorto the mixing. In some embodiments, the mRNA and the pre-formed emptylipid nanoparticle solution are mixed at the ambient temperature butthen heated to the pre-determined temperature after the mixing. In someembodiments, the pre-formed empty lipid nanoparticle solution is heatedto the pre-determined temperature and mixed with mRNA at the ambienttemperature. In some embodiments, the mRNA solution is heated to thepre-determined temperature and mixed with a pre-formed empty lipidnanoparticle solution at ambient temperature.

In some embodiments, the mRNA solution is heated to the pre-determinedtemperature by adding an mRNA stock solution that is at ambienttemperature to a heated buffer solution to achieve the desiredpre-determined temperature.

In some embodiments, the lipid solution containing dissolved lipids, orthe aqueous/buffer solution, or both, may be heated to a pre-determinedtemperature above the ambient temperature prior to mixing. In someembodiments, the lipid solution containing dissolved lipids and theaqueous solution are heated to the pre-determined temperature separatelyprior to the mixing. In some embodiments, the lipid solution containingdissolved lipids and the aqueous solution are mixed at the ambienttemperature but then heated to the pre-determined temperature after themixing. In some embodiments, the lipid solution containing dissolvedlipids is heated to the pre-determined temperature and mixed with anaqueous solution at the ambient temperature. In some embodiments, theaqueous solution is heated to the pre-determined temperature and mixedwith a lipid solution containing dissolved lipids at ambienttemperature. In some embodiments, no heating of one or more of thesolution comprising the pre-formed lipid nanoparticles, the solutioncomprising the mRNA and the mixed solution comprising the lipidnanoparticle encapsulated mRNA occurs before or after the formulationprocess.

In some embodiments, the lipid solution and an aqueous or buffersolution may be mixed using a pump. In some embodiments, an mRNAsolution and a pre-formed empty lipid nanoparticle solution may be mixedusing a pump. As the encapsulation procedure can occur on a wide rangeof scales, different types of pumps may be used to accommodate desiredscale. It is however generally desired to use a pulse-less flow pumps.As used herein, a pulse-less flow pump refers to any pump that canestablish a continuous flow with a stable flow rate. Types of suitablepumps may include, but are not limited to, gear pumps and centrifugalpumps. Exemplary gear pumps include, but are not limited to, Cole-Parmeror Diener gear pumps. Exemplary centrifugal pumps include, but are notlimited to, those manufactured by Grainger or Cole-Parmer.

An mRNA solution and a pre-formed empty lipid nanoparticle solution maybe mixed at various flow rates. Typically, the mRNA solution may bemixed at a rate greater than that of the lipid solution. For example,the mRNA solution may be mixed at a rate at least 1×, 2×, 3×, 4×, 5×,6×, 7×, 8×, 9×, 10×, 15×, or 20× greater than the rate of the lipidsolution.

Suitable flow rates for mixing may be determined based on the scales. Insome embodiments, an mRNA solution is mixed at a flow rate ranging fromabout 40-400 ml/minute, 60-500 ml/minute, 70-600 ml/minute, 80-700ml/minute, 90-800 ml/minute, 100-900 ml/minute, 110-1000 ml/minute,120-1100 ml/minute, 130-1200 ml/minute, 140-1300 ml/minute, 150-1400ml/minute, 160-1500 ml/minute, 170-1600 ml/minute, 180-1700 ml/minute,150-250 ml/minute, 250-500 ml/minute, 500-1000 md/minute, 1000-2000ml/minute, 2000-3000 ml/minute, 3000-4000 ml/minute, or 4000-5000m/minute. In some embodiments, the mRNA solution is mixed at a flow rateof about 200 ml/minute, about 500 mi/minute, about 1000 ml/minute, about2000 ml/minute, about 3000 md/minute, about 4000 ml/minute, or about5000 ml/minute.

In some embodiments, a lipid solution or a pre-formed lipid nanoparticlesolution is mixed at a flow rate ranging from about 25-75 ml/minute,20-50 ml/minute, 25-75 ml/minute, 30-90 ml/minute, 40-100 ml/minute,50-110 mi/minute, 75-200 ml/minute, 200-350 mil/minute, 350-500ml/minute, 500-650 mi/minute, 650-850 mil/minute, or 850-1000 ml/minute.In some embodiments, the lipid solution is mixed at a flow rate of about50 ml/minute, about 100 mil/minute, about 150 m/minute, about 200ml/minute, about 250 ml/minute, about 300 ml/minute, about 350mi/minute, about 400 mil/minute, about 450 ml/minute, about 500ml/minute, about 550 md/minute, about 600 ml/minute, about 650ml/minute, about 700 ml/minute, about 750 ml/minute, about 800ml/minute, about 850 ml/minute, about 900 ml/minute, about 950ml/minute, or about 1000 ml/minute.

Typically, in some embodiments, a lipid solution containing dissolvedlipids, and an aqueous or buffer solution are mixed into a solution suchthat the lipids can form nanoparticles without mRNA (or empty pre-formedlipid nanoparticles). In some embodiments, an mRNA solution and apre-formed lipid nanoparticle solution are mixed into a solution suchthat the mRNA becomes encapsulated in the lipid nanoparticle. Such asolution is also referred to as a formulation or encapsulation solution.A suitable formulation or encapsulation solution includes a solvent suchas ethanol. For example, a suitable formulation or encapsulationsolution includes about 10% ethanol, about 15% ethanol, about 20%ethanol, about 25% ethanol, about 30% ethanol, about 35% ethanol, orabout 40% ethanol.

In some embodiments, a suitable formulation or encapsulation solutionincludes a solvent such as isopropyl alcohol. For example, a suitableformulation or encapsulation solution includes about 10% isopropylalcohol, about 15% isopropyl alcohol, about 20% isopropyl alcohol, about25% isopropyl alcohol, about 30% isopropyl alcohol, about 35% isopropylalcohol, or about 40% isopropyl alcohol.

In some embodiments, a suitable formulation or encapsulation solutionincludes a solvent such as dimethyl sulfoxide. For example, a suitableformulation or encapsulation solution includes about 10% dimethylsulfoxide, about 15% dimethyl sulfoxide, about 20% dimethyl sulfoxide,about 25% dimethyl sulfoxide, about 30% dimethyl sulfoxide, about 35%dimethyl sulfoxide, or about 40% dimethyl sulfoxide.

In some embodiments, a suitable formulation or encapsulation solutionmay also contain a buffering agent or salt. Exemplary buffering agentmay include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate,sodium acetate, potassium phosphate and sodium phosphate. Exemplary saltmay include sodium chloride, magnesium chloride, and potassium chloride.In some embodiments, an empty pre-formed lipid nanoparticle formulationused in making this novel nanoparticle formulation can be stably frozenin 10% trehalose solution.

In some embodiments, an empty (i.e., empty of mRNA) pre-formed lipidnanoparticle formulation used in making this novel nanoparticleformulation can be stably frozen in about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, orabout 50% trehalose solution. In some embodiments, addition of mRNA toempty lipid nanoparticles can result in a final formulation that doesnot require any downstream purification or processing and can be stablystored in frozen form.

In some embodiments, ethanol, citrate buffer, and other destabilizingagents are absent during the addition of mRNA and hence the formulationdoes not require any further downstream processing. In some embodiments,the lipid nanoparticle formulation prepared by this novel processconsists of pre-formed lipid nanoparticles in trehalose solution. Thelack of destabilizing agents and the stability of trehalose solutionincrease the ease of scaling up the formulation and production ofmRNA-encapsulated lipid nanoparticles.

Purification

In some embodiments, the empty pre-formed lipid nanoparticles or thelipid nanoparticles containing mRNA are purified and/or concentrated.Various purification methods may be used. In some embodiments, lipidnanoparticles are purified using Tangential Flow Filtration. Tangentialflow filtration (TFF), also referred to as cross-flow filtration, is atype of filtration wherein the material to be filtered is passedtangentially across a filter rather than through it. In TFF, undesiredpermeate passes through the filter, while the desired retentate passesalong the filter and is collected downstream. It is important to notethat the desired material is typically contained in the retentate inTFF, which is the opposite of what one normally encounters intraditional-dead end filtration.

Depending upon the material to be filtered, TFF is usually used foreither microfiltration or ultrafiltration. Microfiltration is typicallydefined as instances where the filter has a pore size of between 0.05 μmand 1.0 μm, inclusive, while ultrafiltration typically involves filterswith a pore size of less than 0.05 μm. Pore size also determines thenominal molecular weight limits (NMWL), also referred to as themolecular weight cut off (MWCO) for a particular filter, withmicrofiltration membranes typically having NMWLs of greater than 1,000kilodaltons (kDa) and ultrafiltration filters having NMWLs of between 1kDa and 1,000 kDa.

A principal advantage of tangential flow filtration is thatnon-permeable particles that may aggregate in and block the filter(sometimes referred to as “filter cake”) during traditional “dead-end”filtration, are instead carried along the surface of the filter. Thisadvantage allows tangential flow filtration to be widely used inindustrial processes requiring continuous operation since down time issignificantly reduced because filters do not generally need to beremoved and cleaned.

Tangential flow filtration can be used for several purposes includingconcentration and diafiltration, among others. Concentration is aprocess whereby solvent is removed from a solution while solutemolecules are retained. In order to effectively concentrate a sample, amembrane having a NMWL or MWCO that is substantially lower than themolecular weight of the solute molecules to be retained is used.Generally, one of skill may select a filter having a NMWL or MWCO ofthree to six times below the molecular weight of the target molecule(s).

Diafiltration is a fractionation process whereby small undesiredparticles are passed through a filter while larger desired nanoparticlesare maintained in the retentate without changing the concentration ofthose nanoparticles in solution. Diafiltration is often used to removesalts or reaction buffers from a solution. Diafiltration may be eithercontinuous or discontinuous. In continuous diafiltration, adiafiltration solution is added to the sample feed at the same rate thatfiltrate is generated. In discontinuous diafiltration, the solution isfirst diluted and then concentrated back to the starting concentration.Discontinuous diafiltration may be repeated until a desiredconcentration of nanoparticles is reached.

Purified and/or concentrated lipid nanoparticles may be formulated in adesired buffer such as, for example, PBS.

Provided Nanoparticles Encapsulating mRNA

A process according to the present invention results in higher potencyand efficacy thereby allowing for lower doses thereby shifting thetherapeutic index in a positive direction. In some embodiments, theprocess according to the present invention results in homogeneous andsmall particle sizes (e.g., less than 150 nm), as well as significantlyimproved encapsulation efficiency and/or mRNA recovery rate as comparedto a prior art process.

Thus, the present invention provides a composition comprising purifiednanoparticles described herein. In some embodiments, majority ofpurified nanoparticles in a composition, i.e., greater than about 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% ofthe purified nanoparticles, have a size of about 150 nm (e.g., about 145nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm,about 90 nm, about 85 nm, or about 80 nm). In some embodiments,substantially all of the purified nanoparticles have a size of about 150nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

In addition, more homogeneous nanoparticles with narrow particle sizerange are achieved by a process of the present invention. For example,greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% ofthe purified nanoparticles in a composition provided by the presentinvention have a size ranging from about 75-150 nm (e.g., about 75-145nm, about 75-140 nm, about 75-135 nm, about 75-130 nm, about 75-125 nm,about 75-120 nm, about 75-115 nm, about 75-110 nm, about 75-105 nm,about 75-100 nm, about 75-95 nm, about 75-90 nm, or 75-85 nm). In someembodiments, substantially all of the purified nanoparticles have a sizeranging from about 75-150 nm (e.g., about 75-145 nm, about 75-140 nm,about 75-135 nm, about 75-130 nm, about 75-125 nm, about 75-120 nm,about 75-115 nm, about 75-110 nm, about 75-105 nm, about 75-100 nm,about 75-95 nm, about 75-90 nm, or 75-85 nm).

In some embodiments, the dispersity, or measure of heterogeneity in sizeof molecules (PDI), of nanoparticles in a composition provided by thepresent invention is less than about 0.23 (e.g., less than about 0.23,0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11,0.10, 0.09, or 0.08). In a particular embodiment, the PDI is less thanabout 0.16.

In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% of the purified lipid nanoparticles in a compositionprovided by the present invention encapsulate an mRNA within eachindividual particle. In some embodiments, substantially all of thepurified lipid nanoparticles in a composition encapsulate an mRNA withineach individual particle.

In some embodiments, a composition according to the present inventioncontains at least about 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg ofencapsulated mRNA. In some embodiments, a process according to thepresent invention results in greater than about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.

In some embodiments, a composition according to the present invention isformulated so as to administer doses to a subject. In some embodiments,a composition of mRNA lipid nanoparticles as described herein isformulated at a dose concentration of less than 1.0 mg/kg mRNA lipidnanoparticles (e.g., 0.6 mg/kg, 0.5 mg/kg, 0.3 mg/kg, 0.016 mg/kg, 0.05mg/kg, and 0.016 mg/kg. In some embodiments, the dose is decreased dueto the unexpected finding that lower doses yield high potency andefficacy. In some embodiments, the dose is decreased by about 70%, 65%,60%, 55%, 50%, 45% or 40%.

In some embodiments, the potency of mRNA encapsulated lipidnanoparticles produced by Process B is from more than 100% (i.e., morethan 200%, more than 300%, more than 400%, more than 500%, more than600%, more than 700%, more than 800%, or more than 900%) to more than1000% more potent when prepared by Process B as compared to Process A.

Accordingly, in certain embodiments the present invention provides amethod for producing a therapeutic composition comprising purified mRNAthat encodes a peptide or polypeptide for use in the delivery to ortreatment of a human subject. In some embodiments, therapeuticcomposition comprising purified mRNA is used for delivery in the lung ofa subject or a lung cell. In certain embodiments the present inventionprovides a method for producing a therapeutic composition comprisingpurified mRNA that encodes an endogenous protein which may be deficientor non-functional in a subject. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes an endogenous protein which may bedeficient or non-functional in a subject.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes a peptide or polypeptide for use in the delivery to or treatmentof the lung of a subject or a lung cell. In certain embodiments thepresent invention is useful in a method for manufacturing mRNA encodingcystic fibrosis transmembrane conductance regulator, CFTR. The CFTR mRNAis delivered to the lung of a subject in need in a therapeuticcomposition for treating cystic fibrosis. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes a peptide orpolypeptide for use in the delivery to or treatment of the liver of asubject or a liver cell. Such peptides and polypeptides can includethose associated with a urea cycle disorder, associated with a lysosomalstorage disorder, with a glycogen storage disorder, associated with anamino acid metabolism disorder, associated with a lipid metabolism orfibrotic disorder, associated with methylmalonic acidemia, or associatedwith any other metabolic disorder for which delivery to or treatment ofthe liver or a liver cell with enriched full-length mRNA providestherapeutic benefit.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for a protein associated with a urea cycle disorder. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes forornithine transcarbamylase (OTC) protein. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for arginosuccinatesynthetase 1 protein. In certain embodiments the present inventionprovides a method for producing a therapeutic composition comprisingpurified mRNA that encodes for carbamoyl phosphate synthetase 1 protein.In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for arginosuccinate lyase protein. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for arginase protein.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for a protein associated with a lysosomal storage disorder. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for alpha galactosidase protein. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for glucocerebrosidaseprotein. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for iduronate-2-sulfatase protein. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for iduronidaseprotein. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for N-acetyl-alpha-D-glucosaminide protein. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes forheparan N-sulfatase protein. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for galactosamine-6 sulfataseprotein. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for beta-galactosidase protein. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for lysosomal lipaseprotein. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein.In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for transcription factor EB (TFEB).

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for a protein associated with a glycogen storage disorder. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for acid alpha-glucosidase protein. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes forglucose-6-phosphatase (G6PC) protein. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for liver glycogen phosphorylaseprotein. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for muscle phosphoglycerate mutase protein. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes forglycogen debranching enzyme.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for a protein associated with amino acid metabolism. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes forphenylalanine hydroxylase enzyme. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for glutaryl-CoA dehydrogenaseenzyme. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for propionyl-CoA carboxylase enzyme. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for oxalasealanine-glyoxylate aminotransferase enzyme.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for a protein associated with a lipid metabolism or fibroticdisorder. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for an mTOR inhibitor. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for ATPase phospholipidtransporting 8B1 (ATP8B1) protein. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for one or more NF-kappa Binhibitors, such as one or more of I-kappa B alpha, interferon-relateddevelopment regulator 1 (IFRD1), and Sirtuin 1 (SIRT1). In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes forPPAR-gamma protein or an active variant.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for a protein associated with methylmalonic acidemia. Forexample, in certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for methylmalonyl CoA mutase protein. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for methylmalonyl CoAepimerase protein.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA for whichdelivery to or treatment of the liver can provide therapeutic benefit.In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for ATP7B protein, also known as Wilson disease protein. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for porphobilinogen deaminase enzyme. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for one or clottingenzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for human hemochromatosis (HFE) protein.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes a peptide or polypeptide for use in the delivery to or treatmentof the cardiovasculature of a subject or a cardiovascular cell. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for vascular endothelial growth factor A protein. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes forrelaxin protein. In certain embodiments the present invention provides amethod for producing a therapeutic composition comprising purified mRNAthat encodes for bone morphogenetic protein-9 protein. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes for bonemorphogenetic protein-2 receptor protein.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes a peptide or polypeptide for use in the delivery to or treatmentof the muscle of a subject or a muscle cell. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for dystrophinprotein. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for frataxin protein. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes a peptide or polypeptide for usein the delivery to or treatment of the cardiac muscle of a subject or acardiac muscle cell. In certain embodiments the present inventionprovides a method for producing a therapeutic composition comprisingpurified mRNA that encodes for a protein that modulates one or both of apotassium channel and a sodium channel in muscle tissue or in a musclecell. In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for a protein that modulates a Kv7.1 channel in muscle tissue orin a muscle cell. In certain embodiments the present invention providesa method for producing a therapeutic composition comprising purifiedmRNA that encodes for a protein that modulates a Nav1.5 channel inmuscle tissue or in a muscle cell.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes a peptide or polypeptide for use in the delivery to or treatmentof the nervous system of a subject or a nervous system cell. Forexample, in certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for survival motor neuron 1 protein. For example, in certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes forsurvival motor neuron 2 protein. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for frataxin protein. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes for ATPbinding cassette subfamily D member 1 (ABCD1) protein. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes for CLN3protein.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes a peptide or polypeptide for use in the delivery to or treatmentof the blood or bone marrow of a subject or a blood or bone marrow cell.In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for beta globin protein. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for Bruton's tyrosine kinaseprotein. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for one or clotting enzymes, such as Factor VIII, Factor IX,Factor VII, and Factor X.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes a peptide or polypeptide for use in the delivery to or treatmentof the kidney of a subject or a kidney cell. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for collagen type IValpha 5 chain (COL4A5) protein.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes a peptide or polypeptide for use in the delivery to or treatmentof the eye of a subject or an eye cell. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for ATP-bindingcassette sub-family A member 4 (ABCA4) protein. In certain embodimentsthe present invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for retinoschisinprotein. In certain embodiments the present invention provides a methodfor producing a therapeutic composition comprising purified mRNA thatencodes for retinal pigment epithelium-specific 65 kDa (RPE65) protein.In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for centrosomal protein of 290 kDa (CEP290).

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes a peptide or polypeptide for use in the delivery of or treatmentwith a vaccine for a subject or a cell of a subject. For example, incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for an antigen from an infectious agent, such as a virus. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for an antigen from influenza virus. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for an antigen fromrespiratory syncytial virus. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for an antigen from rabies virus.In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for an antigen from cytomegalovirus. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for an antigen fromrotavirus. In certain embodiments the present invention provides amethod for producing a therapeutic composition comprising purified mRNAthat encodes for an antigen from a hepatitis virus, such as hepatitis Avirus, hepatitis B virus, or hepatitis C virus. In certain embodimentsthe present invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for an antigen fromhuman papillomavirus. In certain embodiments the present inventionprovides a method for producing a therapeutic composition comprisingpurified mRNA that encodes for an antigen from a herpes simplex virus,such as herpes simplex virus 1 or herpes simplex virus 2. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes for anantigen from a human immunodeficiency virus, such as humanimmunodeficiency virus type 1 or human immunodeficiency virus type 2. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for an antigen from a human metapneumovirus. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes for anantigen from a human parainfluenza virus, such as human parainfluenzavirus type 1, human parainfluenza virus type 2, or human parainfluenzavirus type 3. In certain embodiments the present invention provides amethod for producing a therapeutic composition comprising purified mRNAthat encodes for an antigen from malaria virus. In certain embodimentsthe present invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for an antigen fromzika virus. In certain embodiments the present invention provides amethod for producing a therapeutic composition comprising purified mRNAthat encodes for an antigen from chikungunya virus.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for an antigen associated with a cancer of a subject oridentified from a cancer cell of a subject. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for an antigendetermined from a subject's own cancer cell, i.e., to provide apersonalized cancer vaccine. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for an antigen expressed from amutant KRAS gene.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for an antibody. In certain embodiments, the antibody can be abi-specific antibody. In certain embodiments, the antibody can be partof a fusion protein. In certain embodiments the present inventionprovides a method for producing a therapeutic composition comprisingpurified mRNA that encodes for an antibody to OX40. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes for anantibody to VEGF. In certain embodiments the present invention providesa method for producing a therapeutic composition comprising purifiedmRNA that encodes for an antibody to tissue necrosis factor alpha. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for an antibody to CD3. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for an antibody to CD19.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for an immunomodulator. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for Interleukin 12. In certainembodiments the present invention provides a method for producing atherapeutic composition comprising purified mRNA that encodes forInterleukin 23. In certain embodiments the present invention provides amethod for producing a therapeutic composition comprising purified mRNAthat encodes for interleukin 36 gamma. In certain embodiments thepresent invention provides a method for producing a therapeuticcomposition comprising purified mRNA that encodes for a constitutivelyactive variant of one or more stimulator of interferon genes (STING)proteins.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for an endonuclease. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for an RNA-guided DNA endonucleaseprotein, such as Cas 9 protein. In certain embodiments the presentinvention provides a method for producing a therapeutic compositioncomprising purified mRNA that encodes for a meganuclease protein. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for a transcription activator-like effector nuclease protein. Incertain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for a zinc finger nuclease protein.

In certain embodiments the present invention provides a method forproducing a therapeutic composition comprising purified mRNA thatencodes for treating an ocular disease. In some embodiments the methodis used for producing a therapeutic composition comprising purified mRNAencoding retinoschisin.

EXAMPLES

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate theinvention and are not intended to limit the same. While certaincompounds, compositions and methods of the present invention have beendescribed with specificity in accordance with certain embodiments, thefollowing examples serve only to illustrate the invention and are notintended to limit the same.

Materials

The lipid nanoparticle formulations described in the following Examples,unless otherwise specified, contain a multi-component lipid mixture ofvarying ratios employing one or more cationic lipids, helper lipids(e.g., non-cationic lipids and/or cholesterol lipids) and PEGylatedlipids designed to encapsulate various nucleic acid materials, asdiscussed previously. The mRNA described in the following Examples wasmRNA encoding either firefly luciferase (FFL-mRNA) or erythropoietin(EPO-mRNA).

Example 1. Encapsulation of mRNA in Lipid Nanoparticle #1 Having LowPEG-Lipid Using Low Concentrations of Lipid Nanoparticle and/or mRNA

This Example illustrates an improvement to Process B for encapsulatingmRNA in a lipid nanoparticle having a low mole % of PEG-lipid. As usedherein, Process B refers to a process of encapsulating messenger RNA(mRNA) by mixing pre-formed lipid nanoparticles with mRNA, as is furtherdescribed in U.S. Published Patent Application No. US2018153822, whichis herein incorporated by reference for all purposes. A range ofdifferent conditions, such as varying temperatures (i.e., heating or notheating the mixture), buffers, and concentrations, may be employed inProcess B. The exemplary conditions described in this and other examplesare for illustration purposes only.

Briefly, the lipids described in Table 1 below were dissolved in ethanoland citrate buffer and first mixed together at the mole percentagesdescribed in Table 1 in the absence of mRNA, in accordance with ProcessB as described in U.S. Published Patent Application No. US2018153822.The instantaneous mixing of the two streams resulted in the formation ofempty lipid nanoparticles, which was a self-assembly process. Theresultant formulation provided empty lipid nanoparticles in citratebuffer containing alcohol, which was buffer-exchanged (e.g., bytangential flow filtration (TFF)) to provide empty lipid nanoparticlesin a 10% wt/volume trehalose solution buffer.

TABLE 1 Lipid Mole % CCBene 50 DMG-PEG 1.5 DSPC 10 Cholesterol 38.5

As per Process B, the resulting suspension of pre-formed empty lipidnanoparticles then was mixed with a suspension of mRNA. The mixing wasconducted with the pre-formed empty lipid nanoparticle suspension andthe mRNA suspension each at the same volume and at the sameconcentration of 0.5 mg/ml. However, this mixing resulted in substantialprecipitation from the mixture, which typically is not observed when thepercentage of PEG-lipid in the lipid nanoparticle is higher, e.g., above3%.

Surprisingly, when the same pre-formed empty lipid nanoparticlesuspension (as described in Table 1) and the same mRNA suspension thenwere mixed each at the same volume but each at a lower concentration, noprecipitation was observed. In particular, when the same pre-formedempty lipid nanoparticle suspension (as described in Table 1) and thesame mRNA suspension were mixed together each at the same volume buteach at 0.1 mg/ml, no precipitation was observed and, moreover, theresulting mRNA-lipid nanoparticle formulation included other desirablefeatures (average particle diameter=139 nm, a polydispersity index(PDI)=0.068 and % mRNA encapsulation=90%). Further, when the samepre-formed empty lipid nanoparticle suspension (as described in Table 1)and the same mRNA suspension were mixed together each at the same volumebut each at an even lower concentration of 0.05 mg/ml, no precipitationwas observed and the resulting mRNA-lipid nanoparticle formulation hadan average particle diameter=123 nm, PDI=0.091 and % mRNAencapsulation=91%.

Example 2. Encapsulation of mRNA in Lipid Nanoparticle #2 Having LowPEG-Lipid Using Low Concentrations of Lipid Nanoparticle and/or mRNA

This Example is another illustration of an improvement to Process B forencapsulating mRNA in a lipid nanoparticle having a low mole % ofPEG-lipid, where the use of lower concentrations of lipid nanoparticleand mRNA in Process B addresses precipitation observed for lipidnanoparticles comprising a low mole percent of PEG-lipid.

In this Example, the lipids described in Table 2 below were dissolved inethanol and citrate buffer and first mixed together at the molepercentages described in Table 2 in the absence of mRNA and then bufferexchanged, in accordance with Process B and as described in Example 1above.

TABLE 2 Lipid Mole % Target23 40 DMG-PEG 3 DOPE 30 Cholesterol 27

As per Process B, the resulting suspension of pre-formed empty lipidnanoparticles then was mixed with a suspension of mRNA. The mixing wasconducted with the pre-formed empty lipid nanoparticle suspension andthe mRNA suspension each at the same volume and each at the sameconcentration of 0.3 mg/ml. However, this mixing resulted in substantialprecipitation from the mixture, which typically is not observed when thepercentage of PEG-lipid in the lipid nanoparticle is higher.

Surprisingly, when the same the pre-formed empty lipid nanoparticlesuspension (as described in Table 2) and the same mRNA suspension werethen mixed each at the same volume but each at lower concentrations, noprecipitation was observed. In particular, when the same pre-formedempty lipid nanoparticle suspension (as described in Table 2) and thesame mRNA suspension were mixed together each at the same volume buteach at 0.1 mg/ml, no precipitation was observed and, moreover, theresulting mRNA-lipid nanoparticle formulation included the desirablefeatures of average particle diameter=92 nm, PDI=0.105 and % mRNAencapsulation=96%. Further, when the same pre-formed empty lipidnanoparticle suspension (as described in Table 2) and the same mRNAsuspension were mixed together each at the same volume but each at aneven lower concentration of 0.05 mg/ml, no precipitation was observedand the resulting mRNA-lipid nanoparticle formulation had an averageparticle diameter=92 nm, PDI=0.127 and % mRNA encapsulation=95%.

Example 3. Encapsulation of mRNA in Lipid Nanoparticle #3 Having LowPEG-Lipid Using Low Concentrations of Lipid Nanoparticle and/or mRNA

This Example is another illustration of an improvement to Process B forencapsulating mRNA in a lipid nanoparticle having a low mole % ofPEG-lipid, where the use of lower concentrations of lipid nanoparticleand mRNA in Process B addresses precipitation observed for lipidnanoparticles comprising a low mole percent of PEG-lipid.

In this Example, the lipids described in Table 3 below were dissolved inethanol and citrate buffer and first mixed together at the molepercentages described in Table 3 in the absence of mRNA and then bufferexchanged, in accordance with Process B and as described in Example 1above.

TABLE 3 Lipid Mole % ML7 50 DMG-PEG 1.5 DOPE 10 Cholesterol 38.5

As per Process B, the resulting suspension of pre-formed empty lipidnanoparticles then was mixed with a suspension of mRNA. The mixing wasconducted with the pre-formed empty lipid nanoparticle suspension andthe mRNA suspension each at the same volume and each at the sameconcentration of 1.0 mg/ml. However, this mixing resulted in substantialprecipitation from the mixture, which typically is not observed when thepercentage of PEG-lipid in the lipid nanoparticle is higher.

Surprisingly, when the same the pre-formed empty lipid nanoparticlesuspension (as described in Table 3) and the same mRNA suspension werethen mixed each at the same volume but each at lower concentrations, noprecipitation was observed. In particular, when the same pre-formedempty lipid nanoparticle suspension (as described in Table 3) and thesame mRNA suspension were mixed together each at the same volume buteach at a lower concentration of 0.01 mg/ml, no precipitation wasobserved and the resulting mRNA-lipid nanoparticle formulation had anaverage particle diameter=163 nm.

Example 4. Encapsulation of mRNA in Lipid Nanoparticle #4 Using LowConcentrations of Lipid Nanoparticle and/or mRNA

This Example is another illustration of an improvement to Process B forencapsulating mRNA in a lipid nanoparticle, where the use of lowerconcentrations of lipid nanoparticle and mRNA in Process B provides fora smaller lipid nanoparticle size in resulting mRNA-lipid nanoparticleformulation.

In this Example, the lipids described in Table 4 below were dissolved inethanol and citrate buffer and first mixed together at the molepercentages described in Table 4 in the absence of mRNA and then bufferexchanged, in accordance with Process B and as described in Example 1above.

TABLE 4 Lipid Mole % ML7 50 DMG-PEG 2.5 DSPC 10 Cholesterol 37.5

As per Process B, the resulting suspension of pre-formed empty lipidnanoparticles then was mixed with a suspension of mRNA. The mixing wasconducted with the pre-formed empty lipid nanoparticle suspension andthe mRNA suspension each at the same volume and each at the sameconcentration of 1.0 mg/ml. In this Example, this mixing did not resultin substantial precipitation from the mixture but the average diameterof the lipid nanoparticle in the resulting mRNA-lipid nanoparticleformulation was relatively large at 152 nm and had a % encapsulation of92%.

However, surprisingly, when the same pre-formed empty lipid nanoparticlesuspension (as described in Table 4) and the same mRNA suspension werethen mixed each at the same volume but each at a lower concentration,the average diameter of the lipid nanoparticle in the resultingmRNA-lipid nanoparticle formulation was smaller. In particular, when thesame pre-formed empty lipid nanoparticle suspension (as described inTable 4) and the same mRNA suspension were mixed together each at thesame volume but each at a lower concentration of 0.1 mg/ml, theresulting mRNA-lipid nanoparticle formulation had smaller averageparticle diameter of 133 nm and a % mRNA encapsulation=85%.

Taken together, these data in these Examples shows that there can besubstantial advantages in lowering the concentrations of lipidnanoparticle and/or mRNA when using the Process B encapsulation methodas described herein and in U.S. Published Patent Application No.US2018153822. These advantages include prevention or avoidance ofprecipitation or aggregation when using a lipid nanoparticle with a lowmole percent of PEG lipid. The advantages also can include providing asmaller lipid nanoparticle size in the resulting mRNA-lipid nanoparticleformulation.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

1.-32. (canceled)
 33. A process of encapsulating messenger RNA (mRNA) inlipid nanoparticles comprising: mixing a solution comprising pre-formedlipid nanoparticles and mRNA such that lipid nanoparticles encapsulatingmRNA are formed, wherein the pre-formed lipid nanoparticles and the mRNAare present in the solution at a concentration of no greater than 0.5mg/ml, and wherein the pre-formed lipid nanoparticles comprise one ormore cationic lipids, one or more helper lipids, and a PEG-modifiedlipid, wherein said PEG-modified lipid is in an amount no greater than3% of total lipids in the lipid nanoparticles.
 34. The process of claim33, wherein the pre-formed lipid nanoparticles are present at aconcentration no greater than 0.4 mg/ml, 0.3 mg/ml, 0.25 mg/ml, 0.2mg/ml, 0.15 mg/ml, 0.1 mg/ml, 0.05 mg/ml, or 0.01 mg/ml.
 35. The processof claim 33, wherein the mRNA is present in the solution at aconcentration of no greater than 0.4 mg/ml, 0.3 mg/ml, 0.25 mg/ml, 0.2mg/ml, 0.15 mg/ml, 0.1 mg/ml, 0.05 mg/ml, or 0.01 mg/ml.
 36. The processof claim 33, wherein the PEG-modified lipid constitutes less than 2.5%,less than 2%, less than 1.5%, or less than 1% of total lipids in thelipid nanoparticles.
 37. The process of claim 33, wherein thePEG-modified lipid constitutes between 0.1% and 3%, or between 0.75% and2.5%, or between 0.5% and 2% of total lipids in the lipid nanoparticles.38. The process of claim 33, wherein the solution comprising pre-formedlipid nanoparticles and mRNA comprises less than 10 mM citrate.
 39. Theprocess of claim 33, wherein the solution comprising pre-formed lipidnanoparticles and mRNA comprises less than 25% non-aqueous solvent. 40.The process of claim 33, further comprising heating the lipidnanoparticles and mRNA to a temperature greater than ambient temperatureafter the mixing.
 41. The process of claim 33, wherein the mRNA and/orthe pre-formed lipid nanoparticles are heated to a temperature greaterthan ambient temperature prior to the mixing.
 42. The process of claim41, wherein the temperature is or is greater than about 30° C., 37° C.,40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C.
 43. Theprocess of claim 41, wherein the temperature ranges from about 25-70°C., about 30-70° C., about 35-70° C., about 40-70° C., about 45-70° C.about 50-70° C., or about 60-70° C.
 44. The process of claim 43, whereinthe temperature is about 65′ C.
 45. The process of claim 33, wherein thepre-formed lipid nanoparticles are formed by mixing lipids dissolved inethanol with an aqueous solution.
 46. The process of claim 45, whereinthe one or more helper lipids is a non-cationic lipid and/or cholesterollipid.
 47. The process of claim 33, wherein greater than about 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of thepre-formed lipid nanoparticles have a size ranging from 75-150 nm. 48.The process of claim 33, wherein substantially all of the pre-formedlipid nanoparticles have a size ranging from 75-150 nm.
 49. The processof claim 33, wherein the process results in an encapsulation rate ofgreater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
 50. Theprocess of claim 33, wherein the process results in greater than about600%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recoveryof mRNA.
 51. The process of claim 33, wherein the process results in nosubstantial aggregation of lipid nanoparticles.
 52. The process of claim33, wherein the mRNA is modified or unmodified.